Nucleic acids that encode testes specific protease and detect DNA hypomethylated in cancer cells

ABSTRACT

The present invention provides Methyl- (or Mutant-) Differential Display (MDD) methods and nucleic acid probes for detecting mutations and the methylation patterns of nucleic acids. The methods of the present invention are particularly useful for detecting and isolating genomic DNA fragments which are near coding and regulatory regions of genes and which are differentially mutated or methylated relative to the corresponding DNA from normal cells. Genes are frequently not methylated in the cells where they are expressed but are methylated in cell types where they are not expressed. Moreover, tumor cell DNA is frequently methylated to a different extent and in different regions than is the DNA of normal cells. The present invention is used for identifying which regions of the genome are methylated or mutated in different cell types, including cancerous cell types. The present invention is also used for diagnosing whether a tissue sample is cancerous, and whether that cancerous condition is non-metastatic or metastatic.

RELATED U.S. APPLICATION DATA

This application is a continuation-in-part of U.S. Ser. No. 08/657,866,filed May 31, 1996, now U.S. Pat. No. 5,871,917, which is herebyincorporated by reference.

FIELD OF THE INVENTION

The present invention provides Methyl- (or Mutant-) Differential Display(MDD) methods and nucleic acid probes for detecting mutations and/or themethylation patterns of nucleic acids. Genes are frequently notmethylated in the cells where they are expressed but are methylated incell types where they are not expressed. Moreover, tumor cell DNA isfrequently methylated to a different extent and in different regionsthan is the DNA of normal cells. The present invention is used foridentifying which regions of the genome are methylated or mutated indifferent cell types, including cancerous cell types. The presentinvention also is used for identifying whether a tissue sample has amethylation or mutation pattern which is normal or like that of knowncancer cells. The present invention can be used to identify aberrantexpression of known or unknown genes. Such aberrant expression may occurat an incorrect location or an incorrect time, i.e. tissue ordevelopmental specificity is lost. Genes and genomic regions discoveredaccording to the present invention are useful for identifying products,e.g. mRNA, protein or fragments thereof, which are aberrantly expressed.

BACKGROUND OF THE INVENTION

DNA is often methylated in normal mammalian cells. For example, DNA ismethylated to determine whether a given gene will be expressed andwhether the maternal or the paternal allele of that gene will beexpressed. See Melissa Little et al., Methylation and p16: Suppressingthe Suppressor, 1 NATURE MEDICINE 633 (1995). While methylation is knownto occur at CpG sequences, only recent studies indicate that CpNpGsequences may be methylated. Susan J. Clark et al., CpNpG Methylation inMammalian Cells, 10 NATURE GENETICS 20, 20 (1995). Methylation at CpGsites has been much more widely studied and is better understood.

Methylation occurs by enzymatic recognition of CpG and CpNpG sequencesfollowed by placement of a methyl (CH₃) group on the fifth carbon atomof a cytosine base. The enzyme that mediates methylation of CpGdinucleotides, 5-cytosine methyltransferase, is essential for embryonicdevelopment—without it embryos die soon after gastrulation. It is notyet clear whether this enzyme methylates CpNpG sites. Peter W. Laird etal., DNA Methylation and Cancer, 3 HUMAN MOLECULAR GENETICS 1487, 1488(1994).

When a gene has many methylated cytosines it is less likely to beexpressed. K. Willson, 7 TRENDS GENET. 107-109 (1991). Hence, if amaternally-inherited gene is more highly methylated than thepaternally-inherited gene, the paternally-inherited gene will generallygive rise to more gene product. Similarly, when a gene is expressed in atissue-specific manner, that gene will often be unmethylated in thetissues where it is active, but will be highly methylated in the tissueswhere it is inactive. Incorrect methylation is thought to be the causeof some diseases including Beckwith-Wiedemann syndrome and Prader-Willisyndrome. I. Henry et al., 351 NATURE 665, 667 (1991); R. D. Nicholls etal., 342 NATURE 281, 281-85 (1989).

The methylation patterns of DNA from tumor cells are generally differentthan those of normal cells. Laird et al., supra. Tumor cell DNA isgenerally undermethylated relative to normal cell DNA, but selectedregions of the tumor cell genome may be more highly methylated than thesame regions of a normal cell's genome. Hence, detection of alteredmethylation patterns in the DNA of a tissue sample is an indication thatthe tissue is cancerous. For example, the gene for Insulin-Like GrowthFactor 2 (IGF2) is hypomethylated in a number of cancerous tissues, suchas Wilm's Tumors, rhabdomyosarcoma, lung cancer and hepatoblastomas.Rainner et al. 362 NATURE 747-49 (1993); Ogawa, et al., 362 NATURE749-51 (1993); S. Zhan et al., 94 J. CLIN. INVEST. 445-48 (1994); P. V.Pedone et al., 3 HUM. MOL. GENET. 1117-21 (1994); H. Suzuki et al., 7NATURE GENET 432-38 (1994); S. Rainier et al., 55 CANCER RES. 1836-38(1995).

The present invention is directed to a method of detecting differentialmethylation at CpNpG sequences by cutting test and control DNAs with arestriction enzyme that will not cut methylated DNA, and then detectingthe difference in size of the resulting restriction fragments.

While methylation-sensitive restriction enzymes have been used forobserving differential methylation in various cells, no commercialassays exist for use on human samples because differentially methylatedsequences represent such a minute proportion of the human genome thatthey are not readily detected. The human genome is both highly complex,in that it contains a great diversity of DNA sequences, and highlyrepetitive, in that it contains a lot of DNA with very similar oridentical sequences. The high complexity and repetitiveness of human DNAconfounds efforts at detecting and isolating the minute amount ofdifferentially methylated DNA which may be present in a test sample. Thepresent invention remedies this detection problem by providing newprocedures for screening a selected subset of the mammalian genome whichis most likely to contain genetic functions.

The present invention provides techniques for detecting and isolatingdifferentially methylated or mutated segments of DNA which may bepresent in a tissue sample in only minute amounts by using one or morerounds of DNA amplification coupled with subtractive hybridization toidentify such segments of DNA. DNA amplification has been coupled withsubtractive hybridization in the Representational Difference Analysis(RDA) procedures disclosed in U.S. Pat. No. 5,436,142 to Wigler et al.and Nikolai Lisitsyn et al., Cloning the Differences Between Two ComplexGenomes, 259 SCIENCE 946 (1993). However, for the subtractivehybridization step of such RDA procedures to proceed in a reasonabletime and with reasonable efficiency, only a subset of the genome can beexamined. To accomplish this necessary reduction in the complexity ofthe sample DNA, Wigler et al. and Lisitsyn et al. disclose cutting DNAsamples with restriction enzymes that cut infrequently and randomly.However, selection of enzymes which randomly cut the genome means thatthe portion of the genome which is examined is not enriched for anyparticular population of DNA fragments. Thus, when RDA is used, only arandom subset of the human genome, which includes repetitive elements,noncoding regions and other sequences which are generally not ofinterest, can be tested in a single experiment.

In contrast, the present invention is directed to methods which useenzymes that cut frequently and that specifically cut CG-rich regions ofthe genome. These enzymes are chosen because CG-rich regions of thegenome are not evenly distributed in the genome—instead, CG-rich regionsare frequently found near genes, and particularly near the promoterregions of genes. This means that the proportion of the genome that isexamined by the present methods will be enriched for genetically-encodedsequences as well as for regulatory sequences. Moreover, unlike the RDAmethod, the present methods selectively identify regions of the genomewhich are hypomethylated or hypermethylated by using enzymes whichspecifically cut non-methylated CG-rich sequences. The present inventiontherefore represents an improvement over RDA methods because of itsability to select DNA fragments which are likely to be near or to encodegenetic functions.

SUMMARY OF THE INVENTION

The present invention provides probes and methods of detecting whether aCNG triplet is hypomethylated or hypermethylated in a genomic DNApresent in a test sample of cell.

The present invention provides a method of detecting whether a CNGtriplet is hypomethylated or hypermethylated in a genomic DNA present ina test sample of cells which includes:

a) isolating genomic DNA from a control sample of cells and a testsample of cells to generate a control-cell DNA and a test-cell DNA;

b) cleaving the control-cell DNA and the test-cell DNA with a masterrestriction enzyme to generate cleaved control-cell DNA and cleavedtest-cell DNA;

c) preparing a probe from a DNA isolated by the present methods, forexample, a DNA selected from the group consisting of SEQ ID NO:7-10;

d) hybridizing the probe to the cleaved control-cell DNA and the cleavedtest-cell DNA to form a control-hybridization complex and atest-hybridization complex; and

e) observing whether the size of the control-hybridization complex isthe same as the size of the test-hybridization complex;

wherein the master restriction enzyme cleaves a nonmethylated CNG DNAsequence but does not cleave a methylated CNG DNA sequence.

Similarly, the present invention provides a method of detecting whethera DNA site is mutated in a genomic DNA present in a test sample of cellswhich includes steps a) through e) above but where a detectorrestriction enzyme is used instead of the master restriction enzyme,wherein the detector restriction enzyme does not cleave a mutated DNAsite but does cleave a corresponding nonmutated DNA site.

The present invention also provides methods of isolating probes todetect hypomethylation or hypermethylation in a CNG triplet of DNA. Inparticular, the present invention provides a method of isolating a probeto detect hypomethylation in a CNG triplet of DNA which includes:

a) cleaving a tester sample of genomic DNA with both a masterrestriction enzyme and a partner restriction enzyme to generate acleaved tester sample;

b) ligating a first set of adaptors onto master enzyme cut DNA ends ofthe cleaved tester sample to generate a first-tester amplificationtemplate;

c) amplifying the first-tester amplification template to generate afirst-tester amplicon by in vitro DNA amplification using primers thathybridize to the first set of adaptors;

d) cleaving off the first adaptors from the first-tester amplicon andligating a second set of adaptors onto DNA ends of the first-testeramplicon to generate a second-adaptor-tester which has second adaptorends;

e) melting and hybridizing the second-adaptor-tester with about a10-fold to about a 10,000-fold molar excess of a driver DNA to generatea mixture of tester-tester product and tester-driver product;

f) adding nucleotides onto DNA ends present in the mixture to make ablunt-ended tester-tester product and a blunt-ended tester-driverproduct;

g) amplifying the blunt-ended tester-tester product and the blunt-endedtester-driver product by in vitro DNA amplification using primers thathybridize to second adaptor ends to generate a second-tester amplicon;

f) isolating a discrete DNA fragment from the second tester amplicon asa probe to detect the hypomethylation or the hypermethylation in a CNGtriplet of DNA;

wherein the master restriction enzyme cleaves a nonmethylated CNG DNAsequence but does not cleave a methylated CNG DNA sequence;

wherein the partner restriction enzyme cleaves DNA to produce DNAfragments with a complexity of about 5% to about 25% of the genomic DNAin a size range which can be amplified by a DNA amplification enzyme;and

wherein the driver DNA is cut with both the master restriction enzymeand the partner restriction enzyme and amplified using primers thatrecognize DNA ends cut by the master restriction enzyme.

Similarly, when the present invention provides a method of isolating aprobe to detect hypermethylation in a CNG triplet of DNA, normal DNA isused instead of the tester DNA and the normal amplicon is annealed andhybridized with a molar excess of tester driver DNA.

Moreover the present methods are used to isolate probes to detect amutation in a test sample of genomic DNA by using a detector enzymeinstead of a partner restriction enzyme, wherein the detectorrestriction enzyme cleaves a normal DNA site but not a mutant DNA site.

The present invention further provides a kit for detectinghypomethylation in a CNG triplet of DNA which is present in a testtissue sample which includes a DNA or a probe isolated by the methods ofthe present invention, for example, a DNA having SEQ ID NO:7-10.

The present invention provides nucleic acid sequences which aredifferentially methylated in human breast and ovarian cancer tissues, ascompared to normal tissue.

The present invention provides nucleic acid sequences which are usefulas probes for determining the methylation state of genomic DNA sequenceswhich are differentially methylated in tumor tissues.

Similarly, the present invention provides nucleic acid sequences whichare useful as probes for the detection of mRNA expression, as well asoligonucleotide sequences which can be used to amplify and thus detectnormal and aberrant expression of particular mRNA species.

The DNA sequences provided herein may also be expressed in host cellsfor the production of the encoded protein or protein fragments.

The proteins and protein fragments expressed from the nucleotidesequences disclosed herein are useful for the production of monoclonalantibodies. Such monoclonal antibodies have a variety of uses including,but not limited to, detection of normal or aberrant protein expressionin human tissues and fluids.

BRIEF DESCRIPTIONS OF THE DRAWINGS

FIG. 1 provides a schematic diagram of how the MDD technique generallyselects for DNA fragments which have non-methylated CG-rich ends. DriverDNA is isolated from a normal cell or tissue sample of the sametissue-type as the tester cell DNA. Tester DNA is isolated from a cellor tissue sample which likely has a mutation or a difference in theextent to which its DNA is methylated. Tester (FIG. 1a) and driver (FIG.1b) DNAs are cleaved by the master (M) and partner (P) restrictionenzymes. The master enzyme will not cleave DNA at sites that have amethyl group (superscript“m” ) but will cleave at sites which arenon-methylated (superscript m having a line drawn through it). Thepartner enzyme cleaves the DNA into a size and a complexity appropriatefor DNA amplification and subtractive hybridization. After cleavage, asmall target fragment is completely excised from the tester DNA (FIG.1b) but not from the driver DNA (FIG. 1b). Adaptors (m-linkers) whichhybridize only to master-enzyme-cut DNA ends are ligated onto the testerand driver DNA fragments. Unique target DNAs from the tester sample willhave adaptors on both ends. Upon DNA amplification using a primer thatrecognizes only the adaptor sequences, only DNA fragments with adaptorson both ends will be efficiently amplified.

FIG. 2 depicts an agarose gel containing electrophoretically separatedDNA fragments isolated by MDD from malignant B-cells derived fromChronic Lymphocytic Leukemia (CLL) patients. Lane A contains thedifferent products isolated from CLL patient #111. Lane M contains HaeIII 0174 DNA size markers. As illustrated, the present MDD methods giverise to discrete fragments of DNA which can readily be isolated andsubcloned or used as probes for identifying the methylation patterns ofgenomic DNA samples from different cell types.

FIG. 3 depicts a Southern blot of Msp I-digested tester (T) and driver(D) DNA hybridized with the CLL58 probe. The lane labeled “probe #58”contains a clone of the CLL58 probe. The lane labeled “T” containsamplified 2 μg CD5⁺B-cell DNA, whereas the lane “D” contains 2 μgamplified neutrophil DNA. Both T and D DNA samples were isolated fromthe CLL patient #111 and amplified by the MDD method. As a positivecontrol, probe CLL58 hybridizes strongly to itself. Probe CLL58 alsohybridizes strongly with the tester DNA but only weakly with the driveramplicon DNA—this is due to DNA hypomethylation in the tester DNA.

FIG. 4 depicts a Southern blot of Msp I-digested DNA hybridized with theCLL58 probe. The lane labeled “probe #58” contains a clone of the CLL58probe. The lane labeled “T” contains 6 μg of CD5⁺B-cell DNA, whereaslane “N” contains 6 μg of neutrophil DNA, each isolated from the CLLpatient #111. As a positive control, probe CLL58 hybridizes strongly toitself. Probe CLL58 hybridizes with a lower band and several upper bandsin B-cell DNA (lane T), while comparatively hybridizing much less withthe lower band in neutrophil cell DNA (lane N). The lesser hybridizationto the lower band in neutrophil DNA is due to DNA methylation whichprevents complete excision of that fragment. Hence, the CLL58 probeprovides a comparative methyl-differential display in B-cells andneutrophil cells.

FIG. 5 depicts an agarose gel after electrophoresis of the differentfinal products isolated from breast patient#13 (lane A), patient #14(lane B) and breast patient #4 (lane C) using the MDD technique. Lane MWcontains Hae III 0174 DNA size markers.

FIG. 6a depicts a Southern blot of Msp I-digested tester (T) and driver(D) DNA hybridized with the BR50 probe. The lane labeled “probe #50”contains a clone of the BR50 probe. The lane labeled “T” containsamplified breast tumor cell DNA, whereas the lane “D” contains amplifiednormal breast cell DNA. Both T and D DNA samples were isolated from thebreast patient #14 and amplified by the MDD method. As a positivecontrol, probe BR50 hybridizes strongly to itself. Probe BR50 alsohybridizes strongly with the tester DNA but only weakly with the driveramplicon DNA—this is due to DNA hypomethylation in the tester DNA.

FIG. 6b depicts a Southern blot of Msp I-digested tester (T) and driver(D) DNA hybridized with the BR104 probe. The lane labeled “probe #104”contains a clone of the BR104 probe. The lane labeled “T” containsamplified breast tumor cell DNA, whereas the lane “D” contains amplifiednormal breast cell DNA. Both T and D DNA samples were isolated from thebreast patient #13 and amplified by the MDD method. As a positivecontrol, probe BR104 hybridizes strongly to itself. Probe BR104 alsohybridizes strongly with the tester DNA but only weakly with the driveramplicon DNA—this is due to DNA hypomethylation in the tester DNA.

FIG. 6c depicts a Southern blot of Msp I-digested tester (T) and driver(D) DNA hybridized with the BR254 probe. The lane labeled “probe #254”contains a clone of the BR254 probe. The lane labeled “T” containsamplified breast tumor cell DNA, whereas the lane “D” contains amplifiednormal breast cell DNA. Both T and D DNA samples were isolated from thebreast patient #4 and amplified by the MDD method. As a positivecontrol, probe BR254 hybridizes strongly to itself. Probe BR254 alsohybridizes strongly with the tester DNA but only weakly with the driveramplicon DNA—this is due to DNA hypomethylation in the tester DNA.

FIG. 7a depicts a genomic Southern blot of Msp I-digested DNA hybridizedwith a probe BR50 isolated from a breast cancer patient. The lanelabeled “probe #50” contains a clone of the isolated BR50 probe. Thelane labeled “T” contains 6 μg of B-cell DNA, whereas lane “N” contains6 μg of normal breast DNA, each isolated from the breast cancer patient#14. As a positive control, probe BR50 hybridizes strongly to itself.Probe BR50 hybridizes strongly with a lower band and weakly with amedium band in breast tumor DNA (lane T). However, in normal breast cellDNA (lane N), probe BR50 hybridizes much less with the lower band andonly weakly to an upper band. The lesser hybridization to the lower bandin normal breast DNA is due to DNA methylation which prevents completeexcision of that fragment. Hence, the BR50 probe provides a comparativemethyl-differential display in tumor and normal breast cells.

FIG. 7b depicts a genomic Southern blot of Msp I-digested DNA hybridizedwith a probe BR104 isolated from a breast cancer patient. The lanelabeled “probe #104” contains a clone of the isolated BR104 probe. Thelane labeled “T” contains 6 μg of tumor DNA, whereas lane “N” contains 6μg of normal breast cell DNA, each isolated from the breast cancerpatient #13. As a positive control, probe BR104 hybridizes strongly toitself. Probe BR104 hybridizes strongly with a lower band and weaklywith a medium band in tumor DNA (lane T). However, in normal breast cellDNA (lane N), probe BR104 hybridizes lightly with the lower band andmoderately with middle and upper bands. The lesser hybridization to thelower band in normal breast DNA is due to DNA methylation which preventscomplete excision of that fragment. Hence, the BR104 probe provides acomparative methyl-differential display in tumorous and normal breastcells.

FIG. 7c depicts a genomic Southern blot of Msp I-digested DNA hybridizedwith a probe BR254 isolated from a breast cancer patient. The lanelabeled “probe #254” contains a clone of the isolated BR254 probe. Thelane labeled “T” contains 6 μg of B-cell DNA, whereas lane “N” contains6 μg of normal breast cell DNA, each isolated from the breast cancerpatient #4. As a positive control, probe BR254 hybridizes strongly toitself. Probe BR254 hybridizes strongly with a lower band and with anupper band in tumor DNA (lane T). However, in normal breast cell DNA(lane N), probe BR254 hybridizes only with the upper band. The lack ofhybridization to the lower band in normal breast DNA is due to DNAmethylation which prevents excision of that fragment. Hence, the BR254probe provides a comparative methyl-differential display in tumorous andnormal breast cells.

FIG. 8 depicts a Southern blot of Msp I-digested genomic DNA from fivedifferent breast cancer patients which was hybridized with the BR50probe. The number for each patient is listed above the bracket. Thehybridization patterns of tumor (T) and matched normal (N) DNAs fromeach patient are provided for comparison. The lane labeled “probe #50”contains a clone of the isolated BR50 probe. The lane labeled “T”contains 6 μg of DNA isolated from a tumor, whereas lane “N” contains 6μg of DNA from normal breast tissue. As a positive control, probe BR50hybridizes strongly to itself. For each of the five patients, probe BR50hybridizes strongly with a lower band and less strongly with an upperband in the tumor DNA samples (T). However, probe BR50 hybridizes muchless with the lower band in the normal DNA samples (N). The lesserhybridization to the lower band in normal DNA is due to DNA methylationwhich prevents complete excision of that fragment. Hence, the BR50 probeprovides a comparative methyl-differential display in breast tumor andnormal DNA samples.

FIG. 9 depicts a Southern blot of Msp I-digested genomic DNA from fourdifferent breast cancer patients which was hybridized with the BR104probe. The number for each patient is listed above the bracket. Thehybridization patterns of tumor (T) and matched normal (N) DNAs fromeach patient are provided for comparison. The lane labeled “probe #104”contains a clone of the isolated BR104 probe. As a positive control,probe BR104 hybridizes strongly to itself. The lanes labeled “T” contain6 μg of DNA isolated from a breast tumor, whereas the lanes labeled “N”contain 6 μg of DNA from normal breast tissue. For tumor DNA from eachof the four patients, probe BR104 hybridizes with a lower band and amiddle band. In the normal DNA, the probe hybridizes mainly with themiddle band and an upper band. The lesser hybridization to the lowerband in normal DNA is due to DNA methylation which prevents completeexcision of that fragment. Hence, the BR104 probe provides a comparativemethyl-differential display in breast tumor and normal breast DNAsamples.

FIG. 10 depicts a Southern blot of Msp I-digested genomic DNA from fourdifferent breast cancer patients which was hybridized with the BR254probe. The number for each patient is listed above the bracket. Thehybridization patterns of tumor (T) and matched normal (N) DNAs fromeach patient are provided for comparison. The lane labeled “probe #254”contains a clone of the isolated BR254 probe. The lane labeled “T”contains 6 μg of DNA isolated from a breast tumor, whereas lane “N”contains 6 μg of DNA from normal breast tissue. As a positive control,probe BR254 hybridizes strongly to itself. For each of the fourpatients, probe BR254 hybridizes strongly with a lower band and lessstrongly with an upper band in the tumor DNA samples (T). However, probeBR254 hybridizes much less with the lower band in the normal DNA samples(N). The lesser hybridization to the lower band in normal DNA is due toDNA methylation which prevents complete excision of that fragment.Hence, the BR254 probe provides a comparative methyl-differentialdisplay in breast tumor and normal DNA samples.

FIG. 11 depicts an amplification pattern detected by probe BR254 in DNAisolated from a breast tumor biopsy. FIG. 11 provides a Southern blot ofnormal (N) and tumor (T) DNA from breast cancer patient #23 which wasdigested with Msp I and hybridized with the BR254 probe. Both the N andT lanes contain 6 μg of DNA. The lane labeled “probe #254” contains aclone of the isolated BR254 probe, which hybridizes to itself. In thetumor DNA, the BR254 probe hybridizes very, very strongly to a DNAfragment to which it hybridizes only moderately in normal DNA. Thisindicates that the breast tumor DNA of patient #23 has a highlyamplified region of genomic DNA which is not present in her normal DNA.

FIG. 12 depicts a Southern blot of Msp I-digested genomic DNA from fivedifferent ovarian cancer patients which was hybridized with the BR50probe. The number for each patient is listed above the bracket. Thehybridization patterns of 6 μg primary tumor DNA (T or T1), 6 μgmetastatic tumor DNA (Tm) and 6 μg normal ovarian DNA (N) from thesepatients are provided for comparison. The lane labeled “probe #50”contains a clone of the isolated BR50 probe. The lane labeled “T ”contains 6 μg of DNA isolated from a ovarian tumor, whereas lane “N”contains 6 μg of DNA from normal ovarian tissue. As a positive control,probe BR50 hybridizes strongly to itself. In primary tumor DNAs, theprobe predominantly hybridizes with the lower bands, and slightlyhybridizes with the upper bands. In metastatic tumor DNAs, the probeonly hybridizes with the lower bands. In normal DNAs, the probehybridized with the lower bands and the upper bands. The lesserhybridization to the lower band in normal DNA is due to DNA methylationwhich prevents complete excision of that fragment. Hence, the BR50 probeprovides a comparative methyl-differential display in ovarian tumor andnormal DNA samples. The degree of DNA hypomethylation may be related tothe progress of the disease.

FIG. 13 depicts a Southern blot of Msp I-digested genomic DNA from fourdifferent ovarian cancer patients which was hybridized with the BR254probe. The number for each patient is listed above the bracket. Thehybridization patterns of tumor (T) and matched normal (N) DNAs fromeach patient are provided for comparison. The lane labeled “probe #254”contains a clone of the isolated BR254 probe. The lane labeled “T”contains 6 μg of DNA isolated from a ovarian tumor, whereas lane “N”contains 6 μg of DNA from normal ovarian tissue. As a positive control,probe BR254 hybridizes strongly to itself. For each of the fourpatients, probe BR254 hybridizes with a lower band and with an upperband in the tumor DNA samples (T). However, probe BR254 hybridizes onlywith the upper band in the normal DNA samples (N). The lack ofhybridization to the lower band in normal DNA is due to DNA methylationwhich prevents excision of that fragment. Hence, the BR254 probeprovides a comparative methyl-differential display in ovarian tumor andnormal DNA samples.

FIG. 14 depicts a Southern blot of Msp I-digested genomic DNA from sixdifferent colon cancer patients which was hybridized with the BR254probe. The number for each patient is listed above the bracket. Thehybridization patterns of 6 μg tumor (T) and 6 μg matched normal (N)DNAs from each patient are provided for comparison. The lane labeled“probe #254” contains a clone of the isolated BR254 probe as a positivecontrol to show that probe BR254 hybridizes strongly to itself. In thetumor DNA, the probe hybridizes with lower and upper bands with equalintensity. In the normal DNA, the probe hybridizes mainly with the lowerband. Hence, the BR254 probe detects alteration of DNA methylation incolon cancer DNA.

FIG. 15 depicts a Northern blot of mRNA samples from several normalhuman tissues, which was hybridized with probe BR50. For quantitativecomparison, the same membrane was hybridized to a control probe which isexpressed in all tissues. As indicated by the band labeled “TSP50”,probe BR50 hybridizes strongly with testes specific mRNA, but not withmRNA from other sources.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods and probes for detecting andisolating DNA sequences which are mutated or methylated in one tissuetype but not in another. These methods are herein named Methyl- (orMutant-) Differential Display (“MDD”) methods. These MDD methods, andprobes that are isolated by MDD, are useful inter alia for identifyinggenes that are expressed in a tissue-specific manner. Moreover, thepresent MDD methods and probes are useful for detecting cancerous cells,viruses, bacteria, point mutations, amplifications, deletions andgenomic rearrangements.

To detect whether a DNA sequence is differentially methylated, thepresent invention uses methylation-sensitive restriction enzymes tocleave tester and control DNAs. These methylation-sensitive restrictionenzymes will cut their DNA recognition sites when those sites are notmethylated but do not cut the DNA site if it is methylated. Hence, anon-methylated tester DNA will be cut into smaller sizes than amethylated control DNA. Similarly, a hypermethylated tester DNA will notbe cleaved and will give rise to larger fragments than a normallynon-methylated control DNA.

The present invention is particularly useful for detecting and isolatingDNA fragments that are normally methylated but which, for some reason,are non-methylated in a small proportion of cells. Such DNA fragmentsmay normally be methylated for a number of reasons. For example, suchDNA fragments may be normally methylated because they contain, or areassociated with, genes that are rarely expressed, genes that areexpressed only during early development, genes that are expressed inonly certain cell-types, and the like.

While highly expressed genes are frequently unmethylated, those highlyexpressed genes are often not very interesting because they are“housekeeping genes” which contribute to generalized cellular functionsand cellular survival, rather than to the determination of what makesone cell-type different from another. Moreover, highly expressed geneshave generally already been isolated and characterized.

Unlike the housekeeping genes, non-methylated but normally-methylated,DNA fragments are present in just a small proportion of cells.Therefore, it is very difficult to detect or isolate those fragments andto distinguish them from non-methylated housekeeping genes. The presentinvention solves this problem by using a combination of DNAamplification and hybridization/subtraction techniques. Theamplification steps enrich the genomic DNA sample with small DNAfragments that have both 5′ and 3′ ends cut by the methylation-sensitiveenzyme. The hybridization/subtraction steps eliminate fragments encodingnon-methylated housekeeping genes and fragments which are cut on onlyone end by the methylation-sensitive enzyme. Several rounds ofamplification and hybridization/subtraction can be used if a discretenumber of fragments are not isolated with the first round.

The fragments isolated by MDD are cut on both ends by themethylation-sensitive master enzyme because only fragments cut on bothends with the master enzyme are efficiently amplified. This isaccomplished by using primers during DNA amplification that recognizeonly the master-enzyme-cut DNA ends. Such primer specificity is createdby adding adaptors to the DNA ends of a sample cut with both the masterenzyme and a partner restriction enzyme. The adaptors have uniquesequences which hybridize with the DNA amplification primers. Thepartner enzyme is used to generate a mixture of DNA fragments of anappropriate size and complexity for DNA amplification but also creates apopulation of DNA molecules which will not accept the adaptors becausethey have partner-enzyme-cut ends.

Similarly, for detecting and isolating fragments which contain amutation, DNA fragments are cut with a master enzyme and a partner(detector) enzyme, where the partner (detector) enzyme may cut normalDNA but which will not cut mutated DNA. The master enzyme normally usedfor MDD is a methylation-sensitive enzyme which recognizes and cutsCG-rich sites in genomic DNA. However, for detecting and isolatingfragments that contain a mutation, nonmethylation sensitive masterenzymes can also be used. Any restriction enzyme can be used as a masterrestriction enzyme for detecting and isolating mutations so long as itcuts at a different sequence than the detector enzyme. However, a masterenzyme which cuts in CG-rich regions is preferred because it selects forregions associated with genes while the detector enzyme identifies andpermits isolation of the mutation by cutting only the nonmutant DNAfragments

To confirm whether fragments isolated by the present MDD methods candetect differentially methylated or mutated DNA sites, they are testedas probes on genomic DNAs which are cut with methylation-sensitive ormutant-detector enzymes, respectively. If the probe detects a differentsize fragment in DNA which is known, or suspected to be,hypomethylated,hypermethylated or mutated than in normal DNA, then theprobe can detect differentially methylated or mutated DNA sites.

DNA fragments isolated by the present methods can therefore constituteprobes, for example, for detecting hypomethylation, hypermethylation ormutation in DNA fragments which are normally methylated.

In particular,the present invention provides a method of isolatingprobes to detect hypomethylation in a CNG triplet of DNA which includes:

a) cleaving a tester sample of genomic DNA with a master restrictionenzyme and a partner restriction enzyme to generate a cleaved testersample;

b) amplifying the cleaved tester sample using primers that recognize DNAends cut by the master restriction enzyme, to generate a first testeramplicon;

c) melting and hybridizing the first test amplicon with a driver DNA togenerate a tester-tester product and a tester-driver product;

d) adding nucleotides to the ends of the tester-tester product and thetester-driver product to make a blunt-ended tester-tester product and ablunt-ended tester-driver product;

e) amplifying the blunt-ended tester-tester product and the blunt-endedtester-driver product by using primers which recognize onlymaster-enzyme-cut DNA ends, to produce a second tester amplicon;

f) isolating a discrete DNA fragment from the second tester amplicon asa probe to detect hypomethylation a CNG triplet of DNA;

wherein the master restriction enzyme cleaves a nonmethylated CNG DNAsequence but does not cleave a methylated CNG DNA sequence;

wherein the partner restriction enzyme cleaves DNA to produce DNAfragments with a complexity of about 5% to about 25% of the genomic DNAin a size range which can be amplified by a DNA amplification enzyme;and

wherein the driver DNA is cut with both the master restriction enzymeand the partner restriction enzyme, and then amplified by using primersthat recognize DNA ends cut by the master restriction enzyme.

In another embodiment, the master-enzyme-cutDNA ends are distinguishedfrom the partner-enzyme-cutDNA ends by ligating a first set of adaptorsonto master-enzyme-cut DNA ends of the cleaved tester sample to generatea first-tester amplification template. This first-tester amplificationtemplate is then amplified to generate a first-tester amplicon by invitro DNA amplification using primers that hybridize to the first set ofadaptors. Hence, only DNA fragments which were cut by the masterrestriction enzyme will be amplified. The first adaptors are thencleaved off the first-tester amplicon and a second set of adaptors isligated onto master-enzyme-cut DNA ends of the first-tester amplicon togenerate a second-adaptor-tester which has second adaptor ends. Asbefore, this tester DNA is melted and hybridized with a large molarexcess of a driver DNA to generate a mixture of tester-tester productand tester-driver product. The DNA ends of these products are made bluntby adding nucleotides and the blunt-ended tester-driver product isamplified the blunt-ended tester-tester product and the blunt-endedtester-driver product by in vitro DNA amplification using primers thathybridize to second adaptor ends. This generates a second-testeramplicon from which a discrete DNA fragments can be isolated as probesto detect the hypomethylation in a CNG triplet of DNA.

As used herein, hypomethylation means that at least one cytosine in a CGor CNG di- or tri-nucleotide site in genomic DNA of a given cell-typedoes not contain CH₃ at the fifth position of the cytosine base. Celltypes which may have hypomethylated CGs or CCGs include any cell typewhich may be expressing a non-housekeeping function. This includes bothnormal cells that express tissue-specific or cell-type specific geneticfunctions, as well as tumorous, cancerous, and similar cell types.Cancerous cell types and conditions which can be analyzed, diagnosed orused to obtain probes by the present methods include Wilm's cancer,breast cancer, ovarian cancer, colon cancer, kidney cell cancer, livercell cancer, lung cancer, leukemia, rhabdomyosarcoma, sarcoma, andhepatoblastoma.

Genomic DNA samples can be obtained from any mammalian body fluid,secretion, cell-type, or tissue, as well as any cultured cell or tissue.

The present MDD technique involves preparation of a driver DNA and atester DNA. A driver DNA is genomic DNA from a “normal” cell type. Atester DNA is genomic DNA from a “test” cell type which may have mutatedDNA sites, hypermethylated DNA sites or non-methylated DNA sites inplace of the normal DNA at that site. Such normal cell types are cellsof the same tissue-type as the “test” cell type, but which are normallymethylated and are not mutated. Test cell types include cells thatexpress tissue or cell-specific functions, cancer cells, tumor cells andthe like. For example, if breast cancer cells are selected as the testcell type, the normal cell type would be breast cells that are notdiseased or cancerous. Preferably, test and normal cell types areisolated from the same person.

After isolation, the tester DNA and the driver DNA are separately cutwith both the master and partner restriction enzymes. As provided by thepresent invention, the master restriction enzyme cleaves a CNG DNAsequence. The master restriction enzyme produces sticky ends, ratherthan blunt ends, when it cuts. In general, the master enzyme used in thepresent methods cleaves only non-methylated CNG DNA sequences and doesnot cleave methylated CNG DNA sequences. However, for selectedapplications a non-methylation sensitive master enzyme can be used, forexample, for detecting and isolating mutations. For detecting andisolating hypomethylated DNA sites, a methylation-sensitive masterenzyme should be used, for example, the Msp I restriction enzyme whichrecognizes and cleaves DNA at nonmethylated CCGG but will not cleave theCCGG sequence when the outer cytosine is methylated. Master restrictionenzymes contemplated by the present invention include Msp I, BsiS I,Hin2 I and the like. In a preferred embodiment, Msp I is used as themaster restriction enzyme for detecting and isolating a DNA fragmentcontaining a hypomethylated site.

According to the present invention, a partner restriction enzyme cleavesDNA to produce DNA fragments with a complexity of about 5% to about 25%of the genomic DNA in a size range which can be amplified by a DNAamplification enzyme. As used herein, the complexity of mixture of DNAfragments is the number of different DNA fragments within a selectedsize range. When mammalian DNA is used, the present methods caneffectively screen a DNA sample containing about 1% to about 25% of themammalian genome. In a preferred embodiment, a DNA sample has about 5%to about 15% of the total DNA sequences in the mammalian genome. In anespecially preferred embodiment, the DNA sample has about 10% of thetotal DNA sequences in the mammalian genome.

According to the present invention, the size range of DNA fragments in asample of genomic DNA is that size range which can be amplified by invitro DNA amplification. When polymerase chain reaction (“PCR”) is used,that size range is about 2000 base pairs (bp) to about 75 bp. Preferablythe selected size range is about 1500 bp to about 100 bp. Morepreferably the selected size is about 1000 bp to about 150 bp.

As used herein, the preferred size is generated by the action of boththe master and partner restriction enzymes. Hence, if the partnerrestriction enzyme generates a population of genomic DNA fragments whichare about 1000 bp or smaller, but, in combination with the masterrestriction enzyme a population of fragments which are less than 50 bpis generated, a different set of partner and master enzymes ispreferably used.

Also according to the present invention, the partner restriction enzymecleaves DNA to produce ends that are neither homologous norcomplementary to a sticky end produced by the master restriction enzyme.

Any convenient restriction enzyme is used as a partner restrictionenzyme to identify CNG sites that are hypomethylated, so long as theappropriate complexity and size of DNA fragments are generated. Forconvenience, a partner restriction enzyme that operates under the sameconditions as the master restriction enzyme may be used. For example,Mse I is a good partner restriction enzyme which can be used under thesame conditions as the preferred Msp I master restriction enzyme. Whenthe preferred Msp I master restriction enzyme is used, the partnerrestriction enzyme preferably recognizes and cuts a 4-nucleotidesequence. Moreover, in a preferred embodiment, the partner restrictionenzyme cuts an AT-rich site or generates a blunt end to avoid anyligation of adaptors, which are designed to attach to master enzyme cutends, onto the ends generated by the partner enzyme. Preferred partnerrestriction enzymes include Mse I, Sau3 A, Rsa I, TspE I, Mae I, NiaIII, Dpn I and the like.

Cleavage methods and procedures for selecting restriction enzymes forcutting DNA at specific sites are known to the skilled artisan. Forexample, many suppliers of restriction enzymes provide information onconditions and types of DNA sequences cut by specific restrictionenzymes, including New England BioLabs, ProMega Biochem,Boehringer-Mannheim and the like. Sambrook et al. provide a generaldescription of methods for using restriction enzymes and other enzymes.See Sambrook et al., 1989 Molecular Cloning: A Laboratory Manual, Vols.1-3 (Cold Spring Harbor Press, Cold Spring Harbor, N.Y.).

After cleaving the DNA with the master and partner restriction enzymes,a set of adaptors is hybridized and ligated onto the sticky DNA endsproduced by the master restriction enzyme. The adapters are selected sothat they will ligate to the CG-rich ends of the DNA cut by the masterrestriction enzyme but not to the ends of the fragments that were cutwith the partner restriction enzyme. Because only the DNA ends offragments in the tester and driver genomic DNA samples will besingle-stranded, adaptors need not be longer than about 12 nucleotidesto specifically recognize and hybridize to the master enzyme-cut ends.However, the adaptors are chosen not only to ligate to DNA ends cut bythe master restriction enzyme, but also to be a good size and DNAsequence to act as recognition sites for primers used during DNAamplification. Adaptors are chosen so that, when the mixture of masterand partner enzyme cut DNA fragments is amplified using primers thathybridize to those adaptors, only those fragments that have the adaptorand thus were cut with the master restriction enzyme will be amplified.Hence, after hybridization and ligation the adaptor ends should have aunique sequence and be of sufficient length to be a unique recognitionsite for primers selected for in vitro DNA amplification.

Preferred sets of adaptors of the present invention include those of SEQID NO: 1-6. As used herein:

the MSA24 adaptor has SEQ ID NO:1—CTCGTCGTCAGGTCAGTGCTTCAC

the MSA12 adaptor has SEQ ID NO:2—CGGTGAAGCACT

the MSB24 adaptor has SEQ ID NO:3—TAGAGCCACGTAGCTGCTGTAGTC

the MSB12 adaptor has SEQ ID NO:4—CGGACTACAGCA

the MSC24 adaptor has SEQ ID NO:5—ACCGTGGACTGGATAGGTTCAGAC

and the MSC12 adaptor has SEQ ID NO:6—CGGTCTGAACCT.

In one embodiment, the SEQ ID NOS:1 and 2 (MSA24 and MSA12) adaptors aresingle-stranded and are used as the first set of adaptors. Thesesingle-stranded adaptors are annealed and hybridized onto DNA ends cutwith the master restriction enzyme and then ligated to those DNA ends byincubation with ligase. Both the tester and driver DNAs receive thefirst set of adaptors. Thus, the master-cut ends of both the tester anddriver DNAs will have adaptors while DNA ends cut with the partnerenzyme, from either the tester or driver, will have no such adaptors.

The tester and driver samples are then separately amplified by known DNAamplification procedures using primers that hybridize to the adaptors.For example, when the SEQ ID NO:1 and 2 adaptors are used, anoligonucleotide having SEQ ID NO:1 can be used as a primer for DNAamplification of only the master enzyme cut DNA fragments. Such DNAamplification generates a tester amplicon and a driver amplicon. In boththe tester and driver amplicons, only fragments which are cut with themaster restriction enzyme will be amplified.

DNA amplification procedures for use in the present MDD methods includeany in vitro amplification procedure which provides sequence-specificsynthesis of a nucleic acid fragment relative to the complex bulk ofnucleic acid present in a sample. The specificity of the process isdetermined by enzymatic recognition of a specific sequence or by theoligonucleotide primers which are capable of hybridizing only withselected adaptors that are added onto the ends of master restriction cutDNA fragments.

In vitro DNA amplification techniques are known in the art. A review ofsuch techniques can be found in Kwoh et al., 8 Am. Biotechnol. Lab. 14(1990). In vitro nucleic acid amplification techniques includepolymerase chain reaction (PCR), transcription-based amplificationsystem (TAS), self-sustained sequence replication system (3SR), ligationamplification reaction (LAR), ligase-based amplification system (LAS),Qβ RNA replication system and run-off transcription.

PCR is a preferred method for DNA amplification. PCR synthesis of DNAfragments occurs by repeated cycles of heat denaturation of DNAfragments, primer annealing onto the adaptor ends of the master-cut DNAfragment, and primer extension. These cycles can be performed manuallyor, preferably, automatically. Thermal cyclers such as the Perkin-ElmerCetus cycler are specifically designed for automating the PCR process,and are preferred. The number of cycles per round of synthesis can bevaried from 2 to more than 50, and is readily determined by consideringthe source and amount of the nucleic acid template, the desired yieldand the procedure for detection of the synthesized DNA fragment.

PCR techniques and many variations of PCR are known. Basic PCRtechniques are described by Saiki et al. (1988 Science 239:487-491) andby U.S. Pat. Nos. 4,683,195, 4,683,202 and 4,800,159, which areincorporated herein by reference.

The conditions generally required for PCR include temperature, salt,cation, pH and related conditions needed for efficient copying of themaster-cut fragment. PCR conditions include repeated cycles of heatdenaturation (i.e. heating to at least about 95° C.) and incubation at atemperature permitting primer: adaptor hybridization and copying of themaster-cut DNA fragment by the amplification enzyme. Heat stableamplification enzymes like the pwo, Thermus aquaticus or Thermococcuslitoralis DNA polymerases are commercially available which eliminate theneed to add enzyme after each denaturation cycle. The salt, cation, pHand related factors needed for enzymatic amplification activity areavailable from commercial manufacturers of amplification enzymes.

As provided herein an amplification enzyme is any enzyme which can beused for in vitro nucleic acid amplification, e.g. by theabove-described procedures. Such amplification enzymes include pwo,Escherichia coli DNA polymerase I, Klenow fragment of E. coli DNApolymerase I, T4 DNA polymerase, T7 DNA polymerase, Thermus aquaticus(Taq) DNA polymerase, Thermococcus litoralis DNA polymerase, SP6 RNApolymerase, T7 RNA polymerase, T3 RNA polymerase, T4 polynucleotidekinase, Avian Myeloblastosis Virus reverse transcriptase, Moloney MurineLeukemia Virus reverse transcriptase, T4 DNA ligase, E. coli DNA ligaseor Qβ replicase. Preferred amplification enzymes are the pwo and Taqpolymerases. The pwo enzyme is especially preferred because of itsfidelity in replicating DNA.

The length of the primers for use in MDD depends on several factorsincluding the nucleotide sequence and the temperature at which thesenucleic acids are hybridized or used during in vitro nucleic acidamplification. The considerations necessary to determine a preferredlength for an oligonucleotide primer are well known to the skilledartisan. For example, as is known to the skilled artisan, the length ofa short nucleic acid or oligonucleotide can relate to its hybridizationspecificity or selectivity. Because the tester and driver samplescontain a complex mixtures of nucleic acids, primers which are shorterthan about 12 nucleotides may hybridize to more than one site in thetest genomic DNA, and accordingly would not have sufficienthybridization selectivity for amplifing only the master-cut fragments.However, a 12- to 15-nucleotide sequence is generally represented onlyonce in a mammalian genome (Sambrook et al. 1989 Molecular Cloning: ALaboratory Manual, Vol. 2, Cold Spring Harbor Press, Cold Spring Harbor,N.Y.; pp. 11.7-11.8). Accordingly, to eliminate amplification offragments which do not have adaptors, primers are chosen which aregenerally at least about 14 nucleotides long.

Preferably, the present primers are at least 16 nucleotides in length.More preferred primers are at least 17 nucleotides in length (Sambrooket al., pp. 11.7-11.8).

After amplification, the first set of adaptors are removed from the endsof the tester and driver amplicons by cleavage with the masterrestriction enzyme. Cleavage with the master restriction enzymepreserves the original ends of the fragments and removes all DNA whichwas not present in the tester DNA as originally isolated.

A second set of adaptors are ligated to the tester amplicon, but not tothe driver amplicon. This generates a second-adaptor-tester sample. Thesecond set of adaptors is selected so that they do not have the samesequence as the first set of adaptors and so that they hybridize, andhence ligate, only to DNA ends cut by the master restriction enzyme. Thesecond set of adaptors should also form a good recognition site forprimers which are used during DNA amplification.

During DNA amplification, non-methylated DNA fragments which arenormally present in all cell types, and which may not be of interest,will also be amplified because they will have adaptors on their ends. Atleast one round of subtraction/hybridization followed by DNAamplification is used by the present MDD methods to remove thesenonmethylated DNA fragments which likely encode “housekeeping”functions.

In general, the present MDD techniques remove “housekeeping” sequencesby hybridizing the amplified tester DNA with a large molar excess ofamplified driver DNA and amplifying only the tester-tester hybrids whichhave perfectly hybridized ends because those perfectly hybridized endsare the only sites recognized by the primers used for DNA amplification.

In particular, the amplified tester DNA (called the first test ampliconor the second-adaptor-tester)is mixed with a large molar excess ofamplified driver DNA. The mixture is denatured, then renatured. As usedherein, a large molar excess of driver to tester DNA is about a 10-foldto about a 10,000-fold molar excess of driver DNA to tester DNA. In apreferred embodiment the tester DNA is melted and hybridized with abouta 10-fold to about a 1000-fold molar excess of a driver DNA. In a morepreferred embodiment, the tester DNA is melted and hybridized with abouta 100-fold molar excess of a driver DNA. Stringent hybridizationconditions are preferably used during renaturation to maximize theformation of fragments with few mismatches.

The skilled artisan can vary the hybridization conditions used duringMDD to achieve optimal hybridization of unique tester DNA fragments totheir tester homolog. Conditions for achieving optimal hybridization areknown to the skilled artisan and generally include temperature and saltconcentrations permitting selective hybridization between two highlyhomologous DNA fragments, e.g. stringent hybridization conditions.Stringent conditions permit little or no detectable hybridizationbetween mismatched driver or tester fragments, that is between fragmentsthat have dissimilar sequences, particularly at the ends. Hybridizationtechniques are described, for example, in Sambrook et al., 1989Molecular Cloning: A Laboratory Manual, Vols. 1-3 (Cold Spring HarborPress, Cold Spring Harbor, N.Y.).

The ends of the renatured fragments are then filled in using a DNApolymerase, e.g. Taq or pwo polymerase. Such a “filling in” reactionmakes the DNA fragments blunt-ended but does not eliminate anyinternally mismatched or unhybridized regions. To eliminate suchmismatched hybrids, the renatured fragments may be treated with anuclease that will remove single-stranded regions and mismatchednucleotides in the renatured fragments. For example, S1 nuclease, mungbean nuclease and the like can be used to eliminate mismatched hybrids.The nuclease will also cleave off mismatched nucleotides at the ends ofeither the tester or the driver fragment. Hence, mismatched hybrids willhave ends that do not contain second adaptor ends. Only thesingle-stranded tester fragments with second adaptor ends thatfaithfully hybridize to an exact tester homolog will end up with asecond adaptor end.

The subtraction/hybridization mixture is then amplified by in vitro DNAamplification procedures using primers that hybridize to the secondadaptor ends. Hence, only the tester DNA fragments with second adaptorends are amplified. Any tester DNA which hybridized with driver DNA willnot be amplified. A large excess of driver DNA is used to promoteformation of hybrids that are commonly found in both the tester anddriver samples. The result is a second tester amplicon with fragmentswhich were uniquely unmethylated in the original tester DNA sample.

DNA fragments cut on only one end with the master restriction enzyme(“single master-cut fragments”) are eliminated by the present inventionis several ways. First, only one end of those fragments will ligate toan adaptor. This means that, during any amplification step, the singlemaster-cut fragments will not be amplified as quickly as the “doublemaster-cut fragments” which have adaptors at both ends. As is known tothe skilled artisan, fragments which hybridize with amplificationprimers at both ends are amplified geometrically, while DNA fragmentswhich hybridize with amplification primers at only one end are amplifiedarithmetically. Second, during the subtraction/hybridization step thosesingle master-cut fragments may also hybridize to their driver homologsbecause the driver is present in vast molar excess. This imperfecthybrid can be eliminated by treatment with nuclease, so it will not beamplified during the subsequent DNA amplification step.

Several rounds of amplification and subtraction/hybridization can beused to isolate discrete DNA fragments which can be used as probes todetect hypomethylation, hypermethylation and mutation in specificregions of the genome. Discrete DNA fragments isolated by the presentMDD methods which can be used to detect hypomethylation,hypermethylation and mutations in genomic DNA samples from differentcell types include DNA fragments having SEQ ID NO:7-10.

While discrete DNA fragments which detect hypomethylation alsofrequently detect hypermethylation, the MDD method is readily adapted topermit isolation of fragments which will specifically detecthypermethylation in identified sites of the genome. This is accomplishedmerely by using normal cell DNA as the source of DNA for thehypermethylation probe. In this case, the normally unmethylated DNA willbe cut by the methylation-sensitive master enzyme while thehypermethylated test DNA will not be cut. To eliminate DNA fragmentswhich are common to the normal and test DNA samples, the normal cell DNAis hybridized with an excess of test cell DNA (now called “test driverDNA”). Hence, isolation of hypermethylation probes generally involvesthe same manipulations, as used for isolation of hypomethylation probes,but the roles of the normal cell DNA and the test cell DNA are reversed.

Also for detecting and isolating DNA fragments that have ahypermethylated CNG, a methylation-sensitive enzyme can be used as apartner enzyme and a non-methylation sensitive enzyme as a masterenzyme. When this modification is used, the adaptors are still designedto attach to the ends of DNA fragments cut by the non-methylationsensitive master restriction enzyme.

In particular, the present invention provides a method of isolating aprobe to detect hypermethylation in a CNG triplet of DNA which includes:

a) cleaving a normal sample of genomic DNA with a master restrictionenzyme and a partner restriction enzyme to generate a cleaved normalsample;

b) amplifying the cleaved normal sample using primers that recognize DNAends cut by the master restriction enzyme, to generate a first normalamplicon;

c) melting and hybridizing the first normal amplicon with a test driverDNA to generate a normal-normal product and a normal-driver product;

d) adding nucleotides to the ends of the normal-normal product and thenormal-driver product to make a blunt-ended normal-normal product and ablunt-ended normal-driver product;

e) amplifying the blunt-ended normal-normal product and the blunt-endednormal-driver product by using primers which recognize onlymaster-enzyme-cut DNA ends, to produce a second normal amplicon;

f) isolating a discrete DNA fragment from the second normal amplicon asa probe to detect the hypermethylation in a CNG triplet of DNA;

wherein the test driver DNA is cut with both the master restrictionenzyme and the partner restriction enzyme, and then amplified by usingprimers that recognize DNA ends cut by the master restriction enzyme.

The normal sample of genomic DNA is obtained by isolating genomic DNAfrom a “normal” cell type, and the test driver DNA is obtained byisolating genomic DNA from a “test” cell type which may havehypermethylated DNA sites in place of the normal non-methylated DNA atthat site. Here, the test cell type is a cancerous or tumorous celltype.

In a further embodiment, the present MDD techniques can be used toidentify and isolate mutations in genomic DNA and it has particularutility for identifying mutations which are near the promoters or codingregions of genes. Nonmethylation-sensitive master restriction enzymescan be used for isolating mutations. However, the preferred masterenzymes recognize CNG DNA sequences including CpG dinucleotides whichare frequently methylated in human genome. Condensed CpG islands appearin gene promoter regions, or even in the first exons. Because CpGislands appear in gene promoter regions, and in the first few exons ofmany genes, MDD-identified lesions will often be close to genes whichcause disease.

According to the present invention, MDD can be used to isolate probesfor detecting any type of genomic lesion. For example, MDD can be usedto identify fragments which have point mutations, deletions, insertions,amplifications, rearrangements, and other mutations.

MDD is readily adapted for isolating mutations in DNA sequence by usinga partner enzyme, now called a detector enzyme, that cuts normal DNA butwhich will not cut the mutated DNA. For example, a point mutation intester DNA can be identified when a cleavage site recognized by thedetector enzyme is eliminated. When cut with master and detectorrestriction enzymes, a fragment with both ends cleaved by the masterenzyme will be present in the tester DNA. However, the detector enzymecleaves this fragment in the driver DNA, because normal DNA has no pointmutation. As a result, the fragment containing the point mutation willbe isolated after performing the present MDD amplification andsubtraction/hybridization steps. The point mutation can be easilyidentified by comparing the DNA sequence of the isolated mutant fragmentwith the DNA sequence of the homolog isolated from a matched normalgenomic DNA. Moreover, the mutant tester fragment will contain theentire mutation—rather than just part of the mutation as would occur ifa detector enzyme were used that cut the mutation rather than the normalDNA site.

Similarly, MDD is readily adapted for isolating deletion mutations inDNA simply by using tester DNA as the driver DNA and normal DNA as thesource of the intact fragment whose homolog in the tester DNA has adeletion. The deletion mutant fragment can be isolated by virtue of itspartial homology with the normal DNA fragment.

In particular, the present invention provides a method of identifying aprobe to detect a mutation in a tester sample of genomic DNA whichincludes:

a) cleaving a tester sample of genomic DNA with both a masterrestriction enzyme and a detector restriction enzyme to generate acleaved tester sample;

b) ligating a first set of adaptors onto master enzyme cut DNA ends ofthe cleaved tester sample to generate a first-tester amplificationtemplate;

c) amplifying the first-tester amplification template to generate afirst-tester amplicon by in vitro DNA amplification using primers thathybridize to the first set of adaptors;

d) cleaving off the first adaptors from the first-tester amplicon andligating a second set of adaptors onto DNA ends of the first-testeramplicon to generate a second adaptor-tester which has second adaptorends;

e) melting and hybridizing the second-adaptor-tester with about a10-fold to about a 10,000-fold molar excess of a driver DNA to generatea mixture of tester-tester product and tester-driver product;

f) adding nucleotides onto DNA ends present in the mixture to make ablunt-ended tester-tester product and a blunt-ended tester-driverproduct;

g) amplifying the blunt-ended tester-tester product and the blunt-endedtester-driver product by in vitro DNA amplification using primers thathybridize to second adaptor ends to generate a second-tester amplicon;

h) isolating a discrete DNA fragment from the second tester amplicon asa probe to detect a mutation in the tester sample of genomic DNA;

wherein the detector restriction enzyme cleaves a normal DNA site but amutant DNA site to produce DNA fragments with a complexity of about 5%to about 25% of the genomic DNA in a size range which can be amplifiedby a DNA amplification enzyme; and

wherein the driver DNA is cut with both the master restriction enzymeand the detector restriction enzyme and amplified using primers thatrecognize DNA ends cut by the master restriction enzyme.

Probes isolated by the present MDD techniques have at least about 14nucleotides to about 2000 nucleotides.

It is well known in the art to adjust hybridization and wash solutioncontents and temperatures such that stringent hybridization conditionsare obtained. Preferred stringent conditions are those that allow aprobe to hybridize to sequences which are more than 90% complementary tothe probe and not to those which are less than 70% complementary.Stringency depends on such parameters as the size and nucleotide contentof the probe being utilized. See Sambrook et al. and other sources forgeneral descriptions and examples.

For identifying mutations and isolating DNA fragments containing thosemutations, selection of a detector restriction enzyme involvesidentifying an enzyme which will not cut mutated genomic DNA but whichwill cut normal DNA. The skilled artisan can readily perform thisanalysis, for example, by testing a plurality of restriction enzymes inthe present MDD methods.

According to the present invention, a mutation, hypomethylation andhypermethylation at a specific site in genomic DNA can be detected byobserving whether the size or intensity of a DNA fragment cut with amaster or detector restriction enzyme is the same in control and testsamples. This can be done by cutting genomic DNA isolated from controland test tissue samples with the master or detector restriction enzyme,hybridizing a probe to the control and test DNAs and observing whetherthe two hybridization complexes are the same or different sizes orintensities.

In particular, the present invention provides a method of detectingwhether a CNG triplet is hypomethylated or hypermethylated in a genomicDNA present in a test sample of cells which includes:

a) isolating genomic DNA from a control sample of cells and a testsample of cells to generate a control-cell DNA and a test-cell DNA;

b) cleaving the control-cell DNA and the test-cell DNA with a masterrestriction enzyme to generate cleaved control-cell DNA and cleavedtest-cell DNA;

c) preparing a probe from a DNA isolated by the methods of the presentinvention, for example, a DNA selected from the group consisting of SEQID NO:7-10;

d) hybridizing the probe to the cleaved control-cell DNA and the cleavedtest-cell DNA to form a control-hybridization complex and atest-hybridization complex; and

e) observing whether the size of the control-hybridization complex isthe same as the size of the test-hybridization complex.

Similarly, the present invention provides a method of detecting whethera DNA site is mutated in a genomic DNA present in a test sample of cellswhich includes:

a) isolating genomic DNA from a control sample of cells and a testsample of cells to generate a control-cell DNA and a test-cell DNA;

b) cleaving the control-cell DNA and the test-cell DNA with a detectorrestriction enzyme to generate cleaved control-cell DNA and cleavedtest-cell DNA;

c) preparing a probe from a discrete DNA isolated by the methods of thepresent invention;

d) hybridizing the probe to the cleaved control-cell DNA and the cleavedtest-cell DNA to form a control-hybridization complex and atest-hybridization complex; and

e) observing whether the size of the control-hybridization complexes thesame as the size of the test-hybridization complex;

wherein the detector restriction enzyme does not cleave a mutated DNAsite but does cleave a corresponding nonmutated DNA site.

Hybridization techniques for detecting specific genomic DNA fragmentsare known in the art and include solid-phase-based hybridization andsolution hybridization which use any of the known reporter molecules.Detailed methodology for gel electrophoretic and nucleic acidhybridization techniques can be found in Sambrook et al.

Preferred nucleic acid probes for detecting hypomethylation andhypermethylation have SEQ ID NOS:7-10.

According to the present invention, a probe isolated by the subjectmethods, can be labeled by any procedure known in the art, for exampleby incorporation of nucleotides linked to a “reporter molecule”.

A “reporter molecule”, as used herein, is a molecule which provides ananalytically identifiable signal allowing detection of a hybridizedprobe. Detection may be either qualitative or quantitative. Commonlyused reporter molecules include fluorophores, enzymes, biotin,chemiluminescentmolecules, bioluminescent molecules, digoxigenin,avidin, streptavidin or radioisotopes. Commonly used enzymes includehorseradish peroxidase, alkaline phosphatase, glucose oxidase andβ-galactosidase, among others. Enzymes can be conjugated to avidin orstreptavidin for use with a biotinylated probe. Similarly, probes can beconjugated to avidin or streptavidin for use with a biotinylated enzyme.The substrates to be used with these enzymes are generally chosen forthe production, upon hydrolysis by the corresponding enzyme, of adetectable color change. For example, p-nitrophenyl phosphate issuitable for use with alkaline phosphatase reporter molecules; forhorseradish peroxidase, 1,2-phenylenediamine, 5-aminosalicylic acid ortolidine are commonly used.

Incorporation of a reporter molecule into a DNA probe can be by anymethod known to the skilled artisan, for example by nick translation,primer extension, random oligo priming, by 3′ or 5′ end labeling or byother means (see, for example, Sambrook et al.).

In another embodiment, the present invention provides one or morecompartmentalized kits for detection of hypomethylation. A first kit hasa receptacle containing at least one of the present isolated probes.Such a probe may be a nucleic acid fragment which is methylated at a CNGtrinucleotide in the genomic DNA of normal cells but which is notmethylated in the genomic DNA of cancerous cells. Such a probe may bespecific for a DNA site that is normally methylated but which isnonmethylated in certain cell types. Similarly, such a probe may bespecific for a DNA site that is abnormally methylated or hypermethylatedin certain cell types. Finally, such a probe may identify a specific DNAmutation. By specific for a DNA site is meant that the probe is capableof hybridizing to the DNA sequence which is mutated or abnormallymethylated, or is capable of hybridizing to DNA sequences adjacent tosaid mutated or abnormally methylated DNA sequences. The probe providedin the present kits may have a covalently attached reporter molecule.

A second kit has at least one receptacle containing a reagent for MDD.Such a reagent can be, for example, a preferred master restrictionenzyme, a preferred partner restriction enzyme, a preferred adaptorwhich can be ligated to DNA cut with the preferred master restrictionenzyme, a primer that hybridizes to a preferred adaptor, and the like.

Using the MDD methods of the present invention, severalmethyl-polymorphic markers or sites in the human genome have beenidentified and DNA fragments encoding these marker sites have beenisolated from different cell types. In particular, the present MDDmethods have been used to identify hypomethylated sites which act asmarkers indicating whether a patient has cancer. These sites wereidentified using B-cells from patients with Chronic Lymphocytic Leukemia(CLL) and using tumor cells from breast cancer patients.

Moreover, for the first time the present methods and probesexperimentally prove that methylated external cytosine at CpCpG tripletsequences do exist in the human genome, and that alteration of theirmethylation patterns is related to human cancer development. Accordingto the present invention, methylation of the first base of CpCpGtriplets is not a random event. Instead, it is a site- andtissue-specific event.

The alteration of the methylation pattern in CpCpG triplets may be akey, and common event, in the development of neoplasia. Imbalancedmethylation at CpCpG triplet may play at least two roles intumorigenesis:

1) DNA hypomethylation may cause some proto-oncogene expression or DNAhypermethylation may silence a tumor supressor which contributes toneoplastic growth; and

2) DNA hypomethylation may change chromatin structure, and induceabnormalities in chromosome pairing and disjunction. Such structuralabnormalities may result in genomic lesions, such as chromosomedeletions, amplifications, inversions, mutations, and translocations,all of which are found in human genetic diseases and cancer.

Hence, the present MDD methods have broad utility for identifyingdifferentially methylated sites at CpCpG triplet sequences in the humangenome; for mapping hypo- and hyper-methylation sites which are relatedto disease development; for understanding the role(s) of DNA methylationin normal cell genomic DNA imprinting, differentiation, and development;for understanding tumorigenesis; for diagnosing and monitoring theprognosis of disease; and for searching for proto-oncogene(s) andrecessive oncogene(s). MDD, as a comprehensive molecular genetictechnique, enables the accumulation of knowledge necessary to helpbetter understand the etiology of human genetic diseases and cancer.

The methods of the present invention permit identification and isolationof genomic nucleic acids which are differentially methylated ordifferentially expressed among tissues of different types. Using thenucleotide sequences provided, oligonucleotides can be constructed whichare useful as reagents for detecting the susceptibility of particularDNA sequences in a test sample to cleavage by restriction endonucleases.Such susceptibility to cleavage is affected by factors which includemethylation of particular bases and the substitution of bases throughmutation. For example, from the sequences provided, it is possible togenerate nucleic acid probes to determine the sizes and relative amountsof particular DNA species which result when DNA recovered from a testsample is digested with selected sequence specific endonucleases.

The genomic nucleic acids isolated by the present invention, oroligonucleotides obtained therefrom, can also be used to detect andisolate expression products, for example, mRNA, complementary DNAs(cDNAs) and proteins derived from or homologous to the present genomicnucleic acids. In one embodiment, the expression of mRNAs homologous tothe nucleic acids of the present invention is detected, for example, byNorthern analysis, reverse transcription or amplification by PCR. Suchprocedures permit detection of mRNAs in a variety of tissue types or atdifferent stages of development. The subject nucleic acids which areexpressed in a tissue-specific or a developmental-stage-specific mannerare useful as tissue-specific markers or for defining the developmentalstage of a sample of cells or tissues. One of skill in the art canreadily adapt the present nucleic acids and oligonucleotides for thesepurposes. Useful hybridization conditions for probes and primers madeaccording to the present invention are readily determinable by those ofskill in the art. Preferred stringent hybridization conditions are thosewhich allow hybridization of nucleic acids which are greater than 90%homologous but which prevent hybridization of nucleic acids which areless than 70% homologous.

In another embodiment of the present invention, cDNAs homologous to thepresent nucleic acids can be isolated by procedures readily available tothose of skill in the art. For example, cDNA libraries can be obtainedfrom the tissues or cells which express the present genomic nucleicacids. Those cDNA libraries can then be probed with a genomic nucleicacid isolated by the methods of the present invention, or anoligonucleotide fragment thereof. Again, one of skill in the art canreadily isolate such cDNAs using the genomic nucleic acids of thepresent invention.

The present genomic and cDNA nucleic acids can be used to prepareoligonucleotides. In one embodiment, oligonucleotides having twelvenucleotides or more can be prepared which hybridize specifically to thepresent genomic nucleic acids and cDNAs and allow detection andisolation of unique nucleic sequences by hybridization. Sequences of atleast 15-20 nucleotides are preferred and are selected from regionswhich lack homology to other known sequences. Sequences of 20 or morenucleotides which lack such homology are optimal.

Genomic nucleic acids isolated by the present procedures include BR50(SEQ ID NO:10), BR104 (SEQ ID NO:7) and BR254 (SEQ ID NO:8), cloneswhich are hypomethylated in breast cancer cells. A cDNA clone identifiedas TSP50 (SEQ ID NO:14) was obtained by virtue of its homology to theBR50 (SEQ ID NO:10) genomic clone. Each of these nucleic acids is usefulfor detecting hypomethylation and expression of homologous nucleic acidsin a variety of normal and cancerous cells or tissues. Each of thesenucleic acids is also useful for identifying homologous genomic and cDNAnucleic acids.

Oligonucleotides useful as probes for screening of samples byhybridization assays or as primers for amplification can be packagedinto kits. Such kits usually contain the probes or primers in apremeasured or predetermined amount, as well as other suitably packagedreagents and materials needed for the particular hybridization oramplification protocol.

In another embodiment, the present genomic and cDNA nucleic acids can beplaced in expression vectors capable of expressing an encoded protein,polypeptide or peptide. Nucleic acids encoding a desired protein orpolypeptide are derived from cDNA or genomic clones using conventionalrestriction digestion or by synthetic methods, and are ligated intovectors from which they may be expressed. Vectors may, if desired,contain nucleic acids encoding portions of other proteins, therebyproviding a fusion protein. For example, portions of the β-galactosidaseor superoxide dismutase coding region may be placed in frame with thepresent coding regions to provide an expression vector which encodes afusion protein.

Expression vectors include plasmids designed for the expression ofproteins or polypeptides fused to or within bacterial phage coatproteins. The DNA encoding the desired protein or polypeptide, whetherin a fusion, premature or mature form, may be ligated into expressionvectors suitable for any host. The DNA encoding the desired polypeptidemay also contain a signal sequence to permit secretion from the intendedhost. Both prokaryotic and eukaryotic host systems are contemplated.

The present proteins, polypeptides and peptides, whether in a premature,mature or fused form, are isolated from lysed cells, or from the culturemedium, and are purified to the extent needed for the intended use. Oneof skill in the art can readily purify these proteins, polypeptides andpeptides by any available procedure. For example, purification may beaccomplished by salt fractionation, size exclusion chromatography, ionexchange chromatography, reverse phase chromatography, affinitychromatography and the like.

The present invention further contemplates antibodies which can bind tothe present proteins, polypeptides and peptides. Such antibodiespreferably bind to unique antigenic regions or epitopes in the presentproteins, polypeptides and peptides. Unique antigenic regions orepitopes of a protein are generally small, often eight to ten aminoacids or less in length. An epitope or antigenic region may be definedby a sequence of as few as 5 amino acids.

Epitopes and antigenic regions useful for generating antibodies can befound within the present proteins, polypeptides or peptides byprocedures available to one of skill in the art. For example, short,unique peptide sequences can be identified in the present proteins andpolypeptides that have little or no homology to known amino acidsequences. Preferably the region of a protein selected to act as apeptide epitope or antigen is not entirely hydrophobic; hydrophilicregions are preferred because those regions likely constitute surfaceepitopes rather than internal regions of the present proteins andpolypeptides. These surface epitopes are more readily detected insamples tested for the presence of the present proteins andpolypeptides.

Peptides can be made by any procedure known to one of skill in the art,for example, by using in vitro translation or chemical synthesisprocedures.

Short peptides which provide an antigenic epitope but which bythemselves are too small to induce an immune response may be conjugatedto a suitable carrier. Suitable carriers and methods of linkage are wellknown in the art. Suitable carriers are typically large macromoleculessuch as proteins, polysaccharides and polymeric amino acids. Examplesinclude serum albumins, keyhole limpet hemocyanin, ovalbumin, polylysineand the like. One of skill in the art can use available procedures andcoupling reagents to link the desired peptide epitope to such a carrier.For example, coupling reagents can be used to form disulfide linkages orthioether linkages from the carrier to the peptide of interest. If thepeptide lacks a disulfide group, one may be provided by the addition ofa cysteine residue. Alternatively, coupling may be accomplished byactivation of carboxyl groups.

The minimum size of peptides useful for obtaining antigen specificantibodies can vary widely. The minimum size must be sufficient toprovide an antigenic epitope which is specific to the protein orpolypeptide. The maximum size is not critical unless it is desired toobtain antibodies to one particular epitope. For example, a largepolypeptide may comprise multiple epitopes, one epitope beingparticularly useful and a second epitope being immunodominant.Typically, antigenic peptides selected from the present proteins andpolypeptides will range from 5 to about 100 amino acids in length. Moretypically, however, such an antigenic peptide will be a maximum of about50 amino acids in length, and preferably a maximum of about 30 aminoacids. It is usually desirable to select a sequence of about 10, 12 or15 amino acids, up to a maximum of about 20 or 25 amino acids.

Amino acid sequences comprising useful epitopes can be identified in anumber of ways. For example, the entire protein sequence can be screenedby preparing a series of peptides that together span the entire proteinsequence. One of skill in the art can routinely test a few largepolypeptides for the presence of an epitope showing a desired reactivityand also test progressively smaller and overlapping fragments toidentify a preferred epitope with the desired specificity andreactivity.

Antigenic polypeptides and peptides are useful for the production ofmonoclonal and polyclonal antibodies. To obtain polyclonal antibodies, aselected animal is immunized with a protein or polypeptide. Serum fromthe animal is collected and treated according to known procedures.Polyclonal antibodies to the protein or polypeptide of interest may thenbe purified by affinity chromatography. Techniques for producingpolyclonal antisera are will known in the art.

Monoclonal antibodies may be made by procedures available to one ofskill in the art, for example, by fusing antibody producing cells withimmortalized cells and thereby making a hybridoma. The generalmethodology for fusion of antibody producing B cells to an immortal cellline is well within the province of one skilled in the art.

The present antibodies are useful for detecting the present proteins andpolypeptides in specific tissues or in body fluids. Moreover, accordingto the present invention, detection of aberrantly expressed proteins orprotein fragments is probative of a disease state. For example,expression of the present proteins or polypeptides from a hypomethylatedgenomic locus may indicate that the protein is being expressed in aninappropriate tissue or at an inappropriate developmental stage. Hence,the present antibodies are useful for detecting diseases associated withprotein expression from improperly methylated genomic loci, for example,the types of cancers and genetic diseases contemplated herein.

Design of immunoassays is subject to a great deal of variation and avariety of these are known in the art. Immunoassays may use a monoclonalor polyclonal antibody reagent which is directed against one epitope ofthe antigen being assayed. Alternatively, a combination of monoclonal orpolyclonal antibodies may be used which are directed against more thanone epitope. Protocols may be based, for example, upon competition,direct antigen-antibody reaction or sandwich type assays. Protocols may,for example, use solid supports or immunoprecipitation. The presentantibodies can be labeled with a reporter molecule for easy detection.Assays which amplify the signal from a bound reagent are also known.Examples include immunoassays which utilize avidin and biotin, or whichutilize enzyme-labeled antibody or antigen conjugates, such as ELISAassays.

Kits suitable for immunodiagnosis and containing the appropriate labeledreagents include antibodies directed against the present proteinepitopes or antigenic regions, packaged appropriately with the remainingreagents and materials required for the conduct of the assay, as well asa suitable set of assay instructions.

The following examples further illustrate the invention.

EXAMPLE 1 Materials and Methods

To test the MDD technique, a working model system was designed toisolate methyl-polymorphic DNA markers (see FIG. 1 for a schematicdiagram of the MDD methods). There is evidence that in mammalian DNA,methylation patterns are both tissue-, and cell-type specific. Hence,DNA was isolated from two different types of cells from patients whohave chronic lymphocytic leukemia (CLL). In CLL patients, the number ofCD5⁺ B type lymphocytes is abnormally high. These CD5⁺ cells can beisolated by highly specific antibodies in a fluorescence activated cellsorter (FACs). For performing MDD, the DNA isolated from these CD5⁺cells served as the tester, while DNA isolated from neutrophils of thesame patient served as the driver. A methyl-polymorphic DNA probe, namedCLL58, which exhibited methyl-differential displays was isolated from anindividual CLL patient.

After the successful isolation of these methyl-polymorphic probes, MDDwas again successfully used in the isolation of methyl-polymorphicprobes from breast cancer biopsies. Three methyl-polymorphic probes,BR50, BR104, and BR254, which gave methyl-differential displays inbreast cancer and their matched normal cells, were isolated from threeindividual patients. BR50 and BR254 also gave methyl-differentialdisplays in ovarian cancer and their matched normal cells, while theBR254 probe exhibited a methyl-differential display in colon cancercells.

DNA Isolation from Malignant B-cells and Neutrophil Cells

Mononuclear and neutrophil cells were isolated from the peripheral wholeblood of CLL patients by Ficoll/Hypage centrifugation. Leukemic B-cells(CD5⁺), were sorted from mononuclear cells in the presence offluorescent anti-CD5 antibodies by FACs. DNAs were isolated by thephenol extraction method. Sambrook, J., Fritsch, E. F. and Maniatis, T.,(1989) Molecular Cloning, 9.16-9.19.

DNA from Human Cancer Biopsies

Dissected human breast, ovarian, and colon cancer tissues (tumor andnormal) were immediately frozen in liquid nitrogen, and stored at −70°C. DNAs were isolated from both tumor and matched normal tissues by thephenol extraction method. Sambrook, J., Fritsch, E. F. and Manitis, T.,(1989) Molecular Cloning, 9.16-9.19.

MDD

To prepare tester and driver amplicons, 1-2 μg of tester and drivergenomic DNA were digested simultaneously with Msp I and Mse Irestriction endonucleases (New England Biolabs). The digested DNAs wereextracted with phenol/chloroform once, and precipitated with 2 volumesof ethanol after adding 1/10 volume of 3 M NaAc.

The DNAs were then resuspended in 10 μl of TE buffer (10 mM Tris-HCl,0.1 mM EDTA, pH8.0). To ligate the first pair of oligonucleotideadapters (MSA24 with SEQ ID NO:1 and MSA12 with SEQ ID NO:2) to thedigested tester and driver amplicon DNAs, 10 μl (1 μg) of digested DNAwas mixed with: 2.5 μl of each of the unphosphorylated single strandadapters having SEQ ID NO:1 (0.6 μg/μl) and SEQ ID NO:2 (0.3 μg/μl); 2μl of 10×ligation buffer (Boehringer Mannheim); and 3 μl of ddH₂O. Theoligonucleotide adapters were annealed by cooling the mix gradually from55° C. to 10° C. over a 1.5 hour period in a 4° C. cold room, and thenligated to Msp I/Mse I-cut DNA fragments by overnight incubation with 1U of T4 DNA ligase (Boehringer Mannheim) at 16° C. After ligation, theligate was diluted with TEt buffer (10 mM Tris-HCl, 0.1 mM EDTA, 20μg/ml of tRNA) at a concentration of 2 ng/μl.

To generate the tester and driver amplicons, tester and driver DNAligated to the first set adapters were separately amplified bypolymerase chain reaction (“PCR”). Two PCR tubes were set up to make thetester amplicon, while five PCR tubes were prepared for the driveramplicon. Each tube has a 400 μl reaction mixture containing 67 mMTris-HCl, pH 8.8 at 25° C., 4 mM MgCl₂, 16 mM (NH₄)₂SO₄, 10 mMβ-mercaptoethanol, 100 μg/ml of non-acetylated bovine serum albumin, 24μl of dNTP (5 mM for each dATP, dCTP, dGTP, and dTTP), 8 μl of SEQ IDNO:1 (0.6 μg/μl), and 80 ng of DNA ligated to the adapters as templates.While the tubes were equilibrated at 72° C. in a thermal cycler(Perkin-Elmer Cetus), 12 U of Taq DNA polymerase (Perkin-Elmer Cetus),and 3 U of pwo DNA polymerase (Boehringer Mannheim) were added. Thereaction was overlaid with mineral oil and incubated for 5 minutes tofill-in the 3′-recessed ends of the ligates, and the DNAs were thenamplified by 24 cycles of PCR. Each PCR cycle included 1 minute at 95°C., 1 minute at 67° C. and 3 minutes at 72° C., and the last cycle wasfollowed with a 10 minute extension time at 72° C. The DNA ampliconswere extracted once with phenol, once with phenol/chloroform, andprecipitated by 2 volumes of 100% ethanol after adding 1/4 volume of 10M NH₄Ac. Amplicon DNAs were then resuspended in TE buffer.

To remove the first set of adapters from the driver amplicon, 80 μg ofdriver amplicon DNA was digested to completion with Msp I enzyme(10U/μg). The driver amplicon DNA was extracted once withphenol/chloroform, and after adding 1/4 volume of 10 M NH₄Ac, the DNAwas precipitated with 2 volumes of 100% ethanol. The amplicon DNA wasresuspended in TE buffer at a concentration of 0.5 μg/μl. To change thetester amplicon DNA adapters, 5 μg of tester amplicon was digested withMsp I (10 U/μg). The digested tester amplicon DNA was purified by PCRpurification spin column (Boebringer Mannheim).

For the hybridization/subtraction and amplification steps, Msp Idigested tester amplicon DNA was ligated to a second set of adapters(MSB24 having SEQ ID NO:3 and MSB12 having SEQ ID NO:4): 1 μl (0.2 μg)of digested tester DNA was mixed with 1 μl of the SEQ ID NO:3 adaptor(0.6 μg/μl), 1 μl of the SEQ ID NO:4 adaptor (0.3 μg/μl), 1 μl of10×ligation buffer, and 6 μl of ddH₂O. The same annealing and ligationsteps were performed as for ligation of the first set of adapters. Afterligation, the ligate was diluted with 40 μl of TEt buffer and mixed with20 μg (40 μl) of driver amplicon DNA. The mix was extracted withphenol/chloroform once, and precipitated by adding 1/4 volume of 10 MNH₄Ac, and 2.5 volumes of 100% ethanol in a dry ice/ethanol bath for 10minutes. The reaction solution was equilibrated to room temperature andthe DNA was collectedly centrifugation. The pellet was washed twice with70% ice cold ethanol, dried, resuspended in 2 μl of 3×EE buffer. D.Straus et al., 87 Proc. Natl. Acad. Sci. USA 1889 (1990). Afterresuspension, the DNA was overlaid with one drop of mineral oil(Perkin-Elmer Cetus), denatured at 100° C. for 5 minutes, and 0.5 μl of5 M NaCl was added. The DNAs were hybridized at 68° C. for at least 20hours.

To amplify the difference products (DP1), the 3′-recessed ends werefirst filled in with DNA polymerase. The hybridized product was dilutedwith 198 μl of TE buffer, and a 50 μl of filling-in reaction was set up:30 μl of diluted DNA hybrids, 10 μl of 5×PCR buffer, 2 μl of dNTP (5 mMfor each dATP, dCTP, dGTP, and dTTP), and 8 μl of ddH₂O. The reactionwas equilibrated at 72° C., 3 U of Taq DNA polymerase (Perkin-ElmerCetus) and 0.75 U of pwo DNA polymerase (Boehringer Mannheim) were addedat 72° C. for 10 minutes. The filled-in DNA hybrids were purified by aPCR purification spin column (Boehringer Mannheim).

To reduce the background for DP1 amplification, mung bean nucleasetreatment of the filled-in DNA hybrids was performed: 9 μl of filled-inDNA hybrid solution mixed with 1 μl of 10×mung bean nuclease reactionbuffer and 50 U of mung bean nuclease (Boehringer Mannheim) wasincubated at 30° C. for 30 minutes. The reaction was terminated by heatinactivation at 100° C. for 5 minutes after adding 40 μl of 50 mMTris-HCl (pH 8.9).

To amplify DP1, 1 tube of 100 μl PCR reaction was set up. The reactionconsisted of: 20 μl of mung bean nuclease-treated DNA hybrid solution,20 μl of 5×PCR buffer, 5 μl of dNTP (5 mM for each dATP, dCTP, dGTP. anddTTP), 2 μl of the SEQ ID NO:3 adaptor, and 52 μl of ddH₂O. The reactionwas hot started at 94° C. for 2 minutes after adding 3 U of Taq DNApolymerase (Perkin-Elmer Cetus) and 0.75 U of pwo DNA polymerase(Boehringer Mannheim). The conditions for the PCR were 1 minute at 95°C., 1 minute at 67° C., and 3 minutes at 72° C. for 35-40 cycles. Thelast cycle was followed with a 10 minutes extension step at 72° C. TheDP1 was purified by PCR purification spin column (Boehringer Mannheim),and the concentration of the DNA was determined at OD₂₆₀ in aspectrophotometer.

To prepare the second round of hybridization/subtraction andamplification steps, 3 μg of DP1 was digested with the restrictionendonuclease Msp I (10 U/μg) in a 30 μl reaction. The digested DP1 waspurified with PCR purification spin column (Beohringer Mannheim).

To put a new set of adapters (MSC24 having SEQ ID NO:5 and MSC12 havingSEQ ID NO:6) on DP1, 2 μl (0.1 μg) of DP1 was mixed with 1 μl of theMSC24 adaptor (0.6 μg/μl), 1 μl of the MSC12 adaptor (0.3 μg/μl), 1 μlof 10×ligation buffer (Beohringer Mannheim), and 6 μl of ddH₂O. Theannealing and ligation reactions were done as before. The ligate wasdiluted with 40 μl of TEt buffer to the concentration of 2 ng/μl.

To set up the hybridization reaction, 20 ng (10 μl) of diluted ligatewas mixed with 20 μg (40 μl) of driver amplicon DNA. The DNA mix wasextracted with an equal volume of phenol/chloroform once, andprecipitated with 2.5 volumes of 100% ethanol after adding 1/4 volume of10 M of NH₄Ac in a dry ice/ethanol bath for 10 minutes. The reactionsolution was equilibrated to room temperature and the DNA was collectedby centrifugation. The pellet was washed twice with 70% ice coldethanol, dried, and resuspended in 2 μl of 3×EE. 0.5 μl of 5 M Nace wasadded after the DNA mix was denatured at 100° C. for 5 minutes. Thehybridization reaction was carried out at 68° C. for a minimum period of20 hours.

To amplify the secondary different product (DP), the hybrids werediluted with 198 μl of TE buffer, and 100 μl of PCR amplificationreaction was set up as follows: 4 μl of the diluted hybrid solution, 5μl of dNTP (5 mM for each dATP, dCTP ,dGTP, dTTP), 20 μl of 5×PCRreaction buffer, and 69 μl of ddH₂O. While the PCR tube was equilibratedat 72° C., 3 U of Taq DNA polymerase (Perkin-Elmer Cetus) and 0.75 U ofpwo DNA polymerase (Boehringer Mannheim) were added to fill-in the3′-recessed ends for 10 minutes. 2 μl of the MSC24 adaptor (0.6 μg/μl)was then added. The PCR reaction was hot started by raising thetemperature to 94° C. for 2 minutes, and 35 to 40 cycles of PCR reactionwere performed under the same conditions as were used for amplifyingDP1.

DP usually contained several individual DNA fragments whenelectrophoresed on a 2% agarose gel. The individual DNA fragments werepurified by DNA gel extraction kit (Qiagen Inc.), and subcloned intopUC118 vector, which was linearized by the restriction endonuclease AccI. If the DP product had some DNA smear, it was treated with mung beannuclease as described above.

For each MDD, twelve to twenty colonies were chosen to amplify theirinserts, from which different sized probes were selected for ampliconSouthern blotting, and human genomic DNA Southern blotting.

Amplicon DNA Southern Blot

The first round of positive probe screening was performed with ampliconDNA Southern blots. Non-Radiation Southern blot and Detection Kits(Genius™) were purchased from Boehringer Mannheim. Probe labeling, anddetection followed the instruction of the manufacturer.

Two to three μg of tester and driver amplicon DNA were electrophoresedon a 2% agarose gel, and blotted to positively charged nylon membranes(Boehringer Mannheim). For prehybridization, the membranes were placedat 68° C. for 2-4 hours in solutions containing 6×SSC, 5×Denhardt'ssolution, 0.5% SDS, 0.1 M EDTA, and 50 μg/ml of salmon sperm DNA. Underthe same conditions, the probes were added, and hybridized to themembranes overnight. The membranes were then rinsed three times with2×SSC, 1×blot wash (12 mM Na₂HPO₄, 8 mM NaH₂PO₄, 1.4 mM Na₄P₂O₇, 0.5%SDS) at 68° C., and further washed three times (30 minutes for eachtime) with the same buffer at 68° C. Next, the membranes wereequilibrated in buffer A (100 mM Tris-HCl, 150 mM, pH 7.5) andtransferred into buffer B (2% black reagent in buffer A) solution andincubated at room temperature for one hour. The membranes were thenwashed 2 times (15 minutes for each time) with buffer A, andequilibrated in buffer C (100 mM Tris-HCl, 100 mM NaCl, 10 MM MgCl₂).Before the membranes were exposed on Kodak X-OMAT film, they were rinsedin lumi-P530 for 1 min and kept in a plastic sheet protector. Positiveclones were identified by observing the probe hybridized more heavily totest amplicon DNA than to driver amplicon DNA.

Human Genomic DNA Southern Blot

The positive probes that were confirmed by the amplicon DNA Southernblot experiment were tested further by human genomic DNA Southernblotting. Genomic DNAs isolated from cancer tissues, and theirrespective matched normal tissues, were digested with Msp I restrictionendonuclease, electrophoresed on 1.5% agarose gels, and transferred toHybond Membranes (Amersham, Arlington Heights, Ill.) These membraneswere exposed to UV light to immobilize the DNA. The probes for theSouthern blot were labeled with High Prime DNA labeling kits (BoehringerMannheim) following the instructions of the manufacturer. The procedurefor hybridization and blot wash were the same as in the Amplicon DNASouthern blotting section.

EXAMPLE 2 Different Methylation Patterns at CpCpG Sites in MalignantB-cells and Neutrophil Cells

DNA isolated from malignant B-cells and neutrophil cells of four CLLpatients (#58, #111, #112, #128) was used to perform MDD as describedabove in the Materials and Methods provided in Example 1. The DNA fromleukemic B-cells was used as the tester, and the DNA from neutrophilcells was used as the driver. Tester and driver DNAs were simultaneouslydigested with the restriction endonucleases, Msp I and Mse I. Afterligating the first set of adapters to the digested tester and driver DNAfragments, tester and driver amplicons were generated by PCRamplification (see Materials and Methods). After two rounds ofhybridization/subtraction and amplification, the difference products(DP) which were isolated from patient 111 appeared as individual DNAfragment bands on an agarose gel (FIG. 2). The DP fragments were thensubcloned into the plasmid pUC118 (see Materials and Methods), andamplified by PCR. For the first round of selection, inserts of differentsizes were selected and hybridized to the tester and driver ampliconDNAs from which they were derived in a non-radioactive Southern blotexperiment (see materials and methods). The positive probes wereselected if they hybridized a single band in the tester amplicon only,or if they hybridized to both tester and driver amplicon DNA, but thehybridization to the driver amplicon DNA band was far less intense. Thefinal proof of a positive probe was that it displayed a differentmethylation pattern in a genomic DNA Southern blot experiment in whichthe parental malignant B-cell, and normal neutrophil cell genomic DNA,were digested with the restriction endonuclease Msp I (see materials andmethods).

The CLL58 probe isolated from patient #111 fulfilled these requirements.When CLL58 was hybridized to its tester and driver amplicon DNA, thetester amplicon DNA gave a heavy hybridization band, while the driveramplicon DNA gave a much less intense hybridized band (FIG.3).

The CLL58 probe was further tested by hybridization to a genomicSouthern blot of Msp I-digested malignant B-cell and neutrophil cellDNA. The genomic Southern blot revealed that a larger fragment was muchless intensely hybridized than a lower molecular weight fragment in themalignant B-cell DNA, while in the neutrophil cell DNA, just theopposite result occurred (FIG. 4). These results demonstrate that inhuman genomic DNA, there are methylated external cytosine residues atCpCpG sequences, and those CpCpG sequences are differentially methylatedin different types of cells. Moreover, the CLL58 probe can detect anddistinguish cell-type specific methylation patterns.

EXAMPLE 3 MDD-Isolated Probes from Human Breast Cancer Tumors alsoDetect Ovarian and Colon Cancers

We have proven the existence of methylation of the external cytosineresidue at CpCpG sites in the human genome, and have isolated CpCpG siterelated methyl-polymorphic markers in a working model system. MDD wasthen applied to successfully isolate CpCpG related methyl-polymorphicmarkers from human breast cancer biopsies.

Isolation of Probes From Breast Tumor DNA

MDD was used to test five pairs of DNA samples (tumor DNA and matchednormal DNA) obtained from five breast cancer patients. To isolatemethyl-polymorphic markers, tumor DNA served as the tester, and normalDNA served as the driver. MDD was performed in the same manner asdescribed in Example 1. Individual DNA fragments were isolated by MDDfrom three breast cancer patients after two rounds ofhybridization/subtraction and amplification (FIG. 5).

Three probes were further analyzed, BR50, BR104, and BR254, isolatedfrom patient #14, patient #13, and patient #4, respectively. Each ofthese three probes appropriately hybridized with their own testeramplicon DNA, but also hybridized slightly with the driver amplicon DNA(FIGS. 6a-c).

These probes were then examined further by hybridizing them to MspI-digested parental tumor and matched normal genomic DNAs. The genomicSouthern blot for probe BR50 showed that the probe hybridized with alower band to a much greater degree than to an upper band in tumor DNA(FIG. 7a). However, in normal DNA, just the opposite occurred—the BR50probe hybridized to the upper band more than to the lower band (FIG.7a).

The genomic DNA Southern blot for probe BR104 showed that the probehybridized with a lower and a middle band in the tumor DNA, while in thenormal DNA, probe BR104 hybridized with a middle and an upper band (FIG.7b).

For the probe BR254, the Southern blot result showed that it hybridizedwith a lower band and a upper band in the tumor DNA, however, it onlyhybridized with an upper band in the normal DNA (FIG. 7c).

The results of three cases, indicate that in normal cells the genomicDNA is more highly methylated, is cut less frequently and gives rise tolarger fragments than is observed for DNA from tumor cells. The isolatedprobes therefore hybridize to higher molecular weight bands in normalDNA. In contrast, the lower bands which were hybridized with probesBR50, BR104, and BR254, are generated by demethylation of the external Cresidue at CpCpG DNA sites in the tumor cells. The weakly hybridizedupper bands in the tumor DNA are likely caused by normal cellcontamination.

Breast Tumor Probes Detect Hypomethylation in Other Cancer Patients

To understand whether this hypomethylation event also happens in otherbreast cancer patients, normal cell and tumor cell DNA was isolated fromten breast cancer patients, cleaved by the Msp I enzyme, and probed withBR50 and BR104. Of the ten breast patients whose DNA was probed withBR50, five had a hybridization pattern which was similar to that of thepatient from which BR50 was isolated (the “parental patient”) (FIG. 8).Of the ten breast patients whose DNA was probed with BR104, five had ahybridization pattern similar to that of the parental patient. (See FIG.9, providing four of these five patterns).

The BR254 probe was hybridized to normal and tumor DNA of 16 breastcancer patients. Of the 16 patients, 10 had a hybridization patternwhich was similar to that of the parental patient. However, somedifferences in the methylation patterns of these patients were observed.Some patients only had the lower band in their tumor DNA, and the upperband in their normal DNA (FIG. 10). However, other patients had thelower band in their tumor DNA, but had both lower and upper bands intheir normal DNA (FIG. 10). These results indicate that the externalcytosine residue at CpCpG sites are fully demethylated in some breasttumors. However, the lower bands appearing in normal DNAs probably werecaused by a partial demethylation event, which suggests that the normalcells were under the process of neoplastic change. In general, thedifferent extent of demethylation identified by probes BR50, BR104, andBR254 in human breast cancer patients is a common phenomena and may beused to predict the extent of neoplastic change.

Detection of a DNA Amplification Event in a Breast Cancer Patient

The BR254 probe detected a highly amplified DNA event in one of the 16breast cancer patients tested with that probe (FIG. 11). This resultprovides evidence that imbalanced DNA methylation may cause aconformational change of chromatin which then induces a DNAamplification.

Hypomethylation Detection by BR50, BR104, and BR254 in Ovarian Cancers.

To examine whether the same hypomethylation phenomena also happens inhuman ovarian cancer, DNA samples isolated from 8 patients with ovariancancer were analyzed. For some patients, two DNA samples were obtained,one from primary ovarian cancer tissue, and the other from their matchednormal tissue. For other patients, three DNA samples were obtained, theywere from primary ovarian cancer tissue, metastatic tumor tissue, andmatched normal tissue. The DNAs were cleaved with Msp I, and hybridizedwith the probes BR50 and BR104 in Southern blot experiments.

Of the eight ovarian patients tested with the BR50 probe, five patientshad similar hybridization patterns: the BR50 probe hybridized almostequally to lower and upper bands in their normal DNA samples, while inthe primary tumor DNA, the upper band was only slightly hybridized.However, in the metastatic tumor DNA of these 5 patients, only the lowerband was hybridized (FIG. 12).

No different hybridization patterns were detected using the probe BR104.This result indicates that the BR104 probe was detecting atissue-specific hypomethylation event.

Eleven patients' DNA samples were examined using the BR254 probe. TheSouthern blot results showed that of 11 patients, 4 had the samehybridization patterns which appeared in the parental DNA pair (FIG.13).

These results suggest that probes BR50 and BR254 detect tumor-relatedhypomethylation events in both breast and ovarian cancer cells.Interestingly, in the case of probe BR50, the complete disappearance ofthe upper band in the metastatic tumor DNA indicates that the extent ofdemethylation seems to be related to the progression of the disease.

Hypomethylation Detection using BR50, BR104, and BR254 on Colon CancerDNA

The methylation patterns of tumor and matched normal DNA samplesisolated from 10 colon cancer patients were examined using BR50, BR104,and BR254 probes. DNAs were digested by the Msp I enzyme, Southern blotsprepared and the blots were hybridized with these probes.

No difference in the methylation patterns of colon tumor DNA and normalDNA were detected by probes BR50 and BR104 (data not shown), indicatingthat both probes were detecting a tissue-specific hypomethylation eventin colon cells.

However, for the probe BR254, different methylation patterns wereobserved. Of the ten colon cancer patients tested, six hadhypomethylation patterns opposite to those observed for the breast andovarian cancer patients. Instead of hybridizing more to an upper band inthe normal DNA samples, probe BR254 hybridized with the lower band.Moreover, instead of hybridizing more to the lower band in the tumorDNA, BR254 hybridized equally with the lower band and a upper band (FIG.14). These results demonstrate that while the BR254 probe detectshypomethylation in breast cancer cells, in colon cancer DNA the BR254probe detects hypermethylation. Therefore, methylation patterns are notonly tissue-type specific, but cancer-specific, and can be different indifferent types of cancers.

Sequencing and Chromosome Assignment of Probes BR50 and BR254

DNA sequencing showed that BR50 contains 1005 bp, with a CG content of58% (SEQ ID NO:10); and BR254 contains 332 bp, with a CG content of 55%(SEQ ID NO:8). Both probes have the nucleotide sequence GGCCGG at oneend, and the nucleotide sequence CCGG sequence at the other end. Msp I,which cleaves at CCGG sequences, is sensitive to methylation at theexternal C residue, and will not cleave CmCGG. Thus, CCGG sequences atthe ends of both probes were methylated in normal breast DNA andhypomethylated in tumor DNA.

Probes BR50 and BR254 were assigned to chromosome #3 and #8 respectivelyby genomic Southern blotting of the Hind III digested monochromosonalhuman/rodent somatic cell hybrid mapping panel #2 (NIGMS Human GeneticMutant Cell Repository). showed that they are located on chromosome #3,and #8, respectively. Fine chromosome mapping was performed usingGeneBridge 4 Radiation Hybrid Panel (Research Genetics, Inc.) astemplates. Walter, M. A. et al., 7 NATURE GENETICS 22 (1994). BR50 isproximally located on 3p12-14, and BR254 is proximally located on8q11-12. The adjacent Sequence Tagged Site (STS) to BR50 is AFMB362WB9,and to BR254 is WI-3862. A known oncogene, Fibroblast Growth FactorReceptor (FGFR) has been located on 8q11-12 region, and our results didshow that the genomic region corresponding to BR254 was amplified in abreast cancer biopsy.

Determination of Coding Sequences

2×10⁶ plaques from a human placenta genomic phage library, EMBL3 SP6/7(ClonTech, Inc.) were screened with the probe BR50. A phage clonehybridizing to BR50 was obtained. The cloned genomic DNA was released bydigestion with Sst I and transferred into pUC118. 8 DNA fragments weregenerated, and the total length of these fragments was approximately 17kbp. Plasmids containing fragments smaller than 1 kbp were completelysequenced, while plasmids containing fragments larger than 1 kbp werepartially sequenced. Homology searches against NIH GenBank revealed twoexons, designated BR50-44 and BR50-45, consisting respectively of 112and 132 nucleotides. Both regions displayed homology to severalmammalian proteases, such as serine proteases and tryptases. Based onthe sequence information obtained, oligomers were designed for thepurpose of amplifying a cDNA sequence from a human universal cDNAlibrary panel (ClonTech Inc.) A PCR product of about 682 bp wasgenerated using one oligomer (SEQ ID NO:11) based on the sequenceobtained from BR50-44 and a second oligomer (SEQ ID NO:12) based on thesequence obtained from BR50-45. This PCR product was directly sequenced.We obtained an incomplete coding sequence of 854 bp by combining thesequence obtained for the PCR product with sequences from BR50-44 andBR50-45. A DNA homology search against NIH GenBank again revealedsimilarity to serine proteases.

Tissue Specific Expression

Two commercially available tissue specific northern blot panels (HumanMultiple Tissue Northern Blot, Human Multiple Tissue Northern Blot II,Clontech Inc.) containing poly A RNA from a total of 16 different humantissues were tested for the expression of an mRNA capable of hybridizingto the coding sequence described above. A probe (SEQ ID NO:13)corresponding to the portion of the coding sequence amplified by theoligomers of SEQ ID NO:11 and SEQ ID NO:12 hybridized strongly andspecifically to a 1.3 kb testes specific transcript. Hybridization tothe remaining 15 human tissues that were tested was not detected (FIG.15). Therefore the locus was designated TSP50 (Testes SpecificProtease).

Isolation of Full Length of cDNA

To obtain a full length TSP50 cDNA, a human testes cDNA A library(Clontech, Inc.) was screened with the TSP50probe having the nucleotidesequence given by SEQ ID NO:13. A cDNA clone consisting of 1,240 bp wasobtained (SEQ ID NO:14). Sequence analysis indicates that this fragmentencodes a protein containing 385 amino acids (SEQ ID NO:16). A stopcodon located 117 bp upstream of the putative translation initiationsite, and a 125 bp 3′ untranslated region before a poly adenosinesequence suggest that a fill length coding sequence has been obtained.

16 1 24 DNA Artificial Sequence Synthetic adapter 1 ctcgtcgtcaggtcagtgct tcac 24 2 12 DNA Artificial Sequence Synthetic adapter 2cggtgaagca ct 12 3 24 DNA Artificial Sequence Synthetic adapter 3tagagccacg tagctgctgt agtc 24 4 12 DNA Artificial Sequence Syntheticadapter 4 cggactacag ca 12 5 24 DNA Artificial Sequence Syntheticadapter 5 accgtggact ggataggttc agac 24 6 12 DNA Artificial SequenceSynthetic adapter 6 cggtctgaac ct 12 7 309 DNA Homo sapiens 7 ccggggccccacaggccccg tgctgaaaca gggctggaac caggcaactt gtttctttgt 60 cctgacatccctgcgcagct ggtaccgagt ggacagcccc aacaaactca tgaacccgct 120 ggtcgctggggtcttcggag ccattgtggg agcggccagt gtcttcggaa atgctcctct 180 gcacgagatcgagccccaga tgcgggacct ggaggtgcac aaatgcataa cacatgggac 240 tgtggctgcaaatcctgagg gaagggcaca agaccttcct acaagggcac tattgcccgc 300 ctgggccgg 3098 332 DNA Homo sapiens 8 ccgggcaatc tccacaggca ctccttccac tgtctgtacaacgtccttag ctcttccttc 60 tgcccagtgt gcaatcttcc cagatgggcc ttctgccacacattcacgtc tgatctggca 120 ggtgtttctt ctgccaaacc ttcatccttc tcggatgctcttctgccaca tgtgcaatcc 180 tgtcagacgt tcctgccact cgaggtagct ggtctgatgtgagcatggaa ccagggggtc 240 cccctaccac caccaggaat gtcagatgat catgaggtgtcggttgggcg gttcttgaac 300 cctatctatg gaggataacc gctcacggcc gg 332 9 568DNA Homo sapiens 9 ccggcctggg aggagggttc ccaccaggct ttctcaagcccatcacaagt gagtcaggaa 60 ccatctccca ttggtcctgg taaggaggag atttggaggaaggactggga agcctgtgga 120 gcgggagggt actgggggtg gaagtggaag aaggtggcacagtaacagac tcccttctcc 180 tgagattccc agcatggatt ggaggggctg ccctgcagcgtcccttaccc cttgttacct 240 ggcagcctgc aagtagctct agggccagcc atgtggtcacctacatctgt tgggggagag 300 aaggaaaaca gatgccccga gtcgtagaga cttgcattatcagcccctgg gtccaacagc 360 aagtgagcta agcacttgca aatgttatct gatgtattctttataagaag gggtcacaaa 420 actgagactt agaacatgta agagggagac agggatttgagcctggatca gctatgtgag 480 gatctcctgt ctgtccccgc tggttccaat tcaggtcttagagtaaaagg gttggggatg 540 agctctctgg caggacagtg ccctccgg 568 10 1005 DNAHomo sapiens 10 ccggaggcag gcacaggact cgggagggac gctgccagct ctctgggtgctgagttcaca 60 aggctgcatt catgattttc aatagacctg tgatggtctg tgcccagtgctggggacaca 120 gaagagtcaa acctggctcc tgacctggac ctggatcatc acgtgacagggaggagagcg 180 atccaggctg atgaggaaag cgcatgacat ggggtcttag gagcagtgaggggcagagcc 240 atggccaaag gccccgccat ggaagctgag gactctggca ccagatggaggcagttgacc 300 gacctctgcc cttggggtcc aacccatggg cttctcatac ataggggtgaaaaaggccat 360 tctatttatg cagaattttc ccatgtggcc aggcagcaga agtccagaggggtaggggcc 420 actcagggtc acacagaaca gcagttgctg aagactgggg aagtccaggcctaggctcca 480 cctgcccttc ccctgacatg gggccaccac tagcctttta tgggcaggcctggctgctgg 540 tggttggaat aacatctgac tccagtgggt gtctgtcacc gtctccagacaggagacaga 600 gacagagggt caaagttcac tatggctctt tggggcaatg aaatgctgtgttctagcctc 660 ttgccagaaa tcagccaaag tcaaggaaag cctgactccc acagttatcacagaaagagc 720 acccactttc cagcccagac agctgcaccc cagctgggtc ctggcagccccagcttcagc 780 ctgggcggta tgttccaggc ccctcgatca tctgacccta atatcaccccttcacacccc 840 ctccactttc tgcgggagcc accccgaacc tttgaatggg ggagatcctggaggctctgc 900 aattttcagt gtaaactgcc tggagttccc cacttcaccc tcatctggttcacctgtgga 960 ctcccaacag agcaggccca ggaaacgcgg ggcctctgag gccgg 1005 1117 DNA Artificial Sequence Synthetic primer 11 cctggatggt cagcgtg 17 1218 DNA Artificial Sequence Synthetic primer 12 cagtgttggt aggaggag 18 13682 DNA Homo Sapiens Synthetic probe 13 cctggatggt cagcgtgcgg gccaatggcacacacatctg tgccggcacc atcattgcct 60 cccagtgggt gctgactgtg gcccactgcctgatctggcg tgatgttatc tactcagtga 120 gggtggggag tccgtggatt gaccagatgacgcagaccgc ctccgatgtc ccggtgctcc 180 aggtcatcat gcatagcagg taccgggcccagcggttctg gtcctgggtg ggccaggcca 240 acgacatcgg cctcctcaag ctcaagcaggaactcaagta cagcaattac gtgcggccca 300 tctgcctgcc tggcacggac tatgtgttgaaggaccattc ccgctgcact gtgacgggct 360 ggggactttc caaggctgac ggcatgtggcctcagttccg gaccattcag gagaaggaag 420 tcatcatcct gaacaacaaa gagtgtgacaatttctacca caacttcacc aaaatcccca 480 ctctggttca gatcatcaag tcccagatgatgtgtgcgga ggacacccac agggagaagt 540 tctgctatga gctaactgga gagcccttggtctgctccat ggagggcacg tggtacctgg 600 tgggattggt gagctggggt gcaggctgccagaagagcga ggccccaccc atctacctac 660 aggtctcctc ctaccaacac tg 682 141240 DNA Homo sapiens CDS 60..1214 cDNA 14 gtcgtggggg cggcactgggagcgccttcc ggagagacgc agtcggctgc caccccggg 59 atg ggt cgc tgg tgc cagacc gtc gcg cgc ggg cag cgc ccc cgg acg 107 Met Gly Arg Trp Cys Gln ThrVal Ala Arg Gly Gln Arg Pro Arg Thr 5 10 15 tct gcc ccc tcc cgc gcc ggtgcc ctg ctg ctg ctg ctt ctg ttg ctg 155 Ser Ala Pro Ser Arg Ala Gly AlaLeu Leu Leu Leu Leu Leu Leu Leu 20 25 30 agg tct gca ggt tgc tgg ggc gcaggg gaa gcc ccg ggg gcg ctg tcc 203 Arg Ser Ala Gly Cys Trp Gly Ala GlyGlu Ala Pro Gly Ala Leu Ser 35 40 45 act gct gat ccc gcc gac cag agc gtccag tgt gtc ccc aag gcc acc 251 Thr Ala Asp Pro Ala Asp Gln Ser Val GlnCys Val Pro Lys Ala Thr 50 55 60 tgt cct tcc agc cgg cct cgc ctt ctc tggcag acc ccg acc acc cag 299 Cys Pro Ser Ser Arg Pro Arg Leu Leu Trp GlnThr Pro Thr Thr Gln 65 70 75 80 aca ctg ccc tcg acc acc atg gag acc caattc cca gtt tct gaa ggc 347 Thr Leu Pro Ser Thr Thr Met Glu Thr Gln PhePro Val Ser Glu Gly 85 90 95 aaa gtc gac cca tac cgc tcc tgt ggc ttt tcctac gag cag gac ccc 395 Lys Val Asp Pro tyr Arg Ser Cys Gly Phe Ser tyrGlu Gln Asp Pro 100 105 110 acc ctc agg gac cca gaa gcc gtg gct cgg cggtgg ccc tgg atg gtc 443 Thr Leu Arg Asp Pro Glu Ala Val Ala Arg Arg TrpPro Trp Met Val 115 120 125 agc gtg cgg gcc aat ggc aca cac atc tgt gccggc acc atc att gcc 491 Ser Val Arg Ala Asn Gly Thr His Ile Cys Ala GlyThr Ile Ile Ala 130 135 140 tcc cag tgg gtg ctg act gtg gcc cac tgc ctgatc tgg cgt gat gtt 539 Ser Gln Trp Val Leu Thr Val Ala His Cys Leu IleTrp Arg Asp Val 145 150 155 160 atc tac tca gtg agg gtg ggg agt ccg tggatt gac cag atg acg cag 587 Ile tyr Ser Val Arg Val Gly Ser Pro Trp IleAsp Gln Met Thr Gln 165 170 175 acc gcc tcc gat gtc ccg gtg ctc cag gtcatc atg cat agc agg tac 635 Thr Ala Ser Asp Val Pro Val Leu Gln Val IleMet His Ser Arg Tyr 180 185 190 cgg gcc cag cgg ttc tgg tcc tgg gtg ggccag gcc aac gac atc ggc 683 Arg Ala Gln Arg Phe Trp Ser Trp Val Gly GlnAla Asn Asp Ile Gly 195 200 205 ctc ctc aag ctc aag cag gaa ctc aag tacagc aat tac gtg cgg ccc 731 Leu Leu Lys Leu Lys Gln Glu Leu Lys tyr SerAsn tyr Val Arg Pro 210 215 220 atc tgc ctg cct ggc acg gac tat gtg ttgaag gac cat tcc cgc tgc 779 Ile Cys Leu Pro Gly Thr Asp tyr Val Leu LysAsp His Ser Arg Cys 225 230 235 240 act gtg acg ggc tgg gga ctt tcc aaggct gac ggc atg tgg cct cag 827 Thr Val Thr Gly Trp Gly Leu Ser Lys AlaAsp Gly Met Trp Pro Gln 245 250 255 ttc cgg acc att cag gag aag gaa gtcatc atc ctg aac aac aaa gag 875 Phe Arg Thr Ile Gln Glu Lys Glu Val IleIle Leu Asn Asn Lys Glu 260 265 270 tgt gac aat ttc tac cac aac ttc accaaa atc ccc act ctg gtt cag 923 Cys Asp Asn Phe tyr His Asn Phe Thr LysIle Pro Thr Leu Val Gln 275 280 285 atc atc aag tcc cag atg atg tgt gcggag gac acc cac agg gag aag 971 Ile Ile Lys Ser Gln Met Met Cys Ala GluAsp Thr His Arg Glu Lys 290 295 300 ttc tgc tat gag cta act gga gag cccttg gtc tgc tcc atg gag ggc 1019 Phe Cys tyr Glu Leu Thr Gly Glu Pro LeuVal Cys Ser Met Glu Gly 305 310 315 320 acg tgg tac ctg gtg gga ttg gtgagc tgg ggt gca ggc tgc cag aag 1067 Thr Trp tyr Leu Val Gly Leu Val SerTrp Gly Ala Gly Cys Gln Lys 325 330 335 agc gag gcc cca ccc atc tac ctacag gtc tcc tcc tac caa cac tgg 1115 Ser Glu Ala Pro Pro Ile tyr Leu GlnVal Ser Ser tyr Gln His Trp 340 345 350 atc tgg gac tgc ctc aac ggg caggcc ctg gcc ctg cca gcc cca tcc 1163 Ile Trp Asp Cys Leu Asn Gly Gln AlaLeu Ala Leu Pro Ala Pro Ser 355 360 365 agg acc ctg ctc ctg gca ctc ccactg ccc ctc agc ctc ctt gct gcc 1211 Arg Thr Leu Leu Leu Ala Leu Pro LeuPro Leu Ser Leu Leu Ala Ala 370 375 380 ctc tgactctgtg tgccctccc tcacttg1240 Leu 385 15 1155 DNA Homo sapiens CDS 1..1155 coding region of cDNA15 tgg gtc gct ggt gcc aga ccg tcg cgc gcg ggc agc gcc ccc gga cgt 48Met Gly Arg Trp Cys Gln Thr Val Ala Arg Gly Gln Arg Pro Arg Thr 5 10 15ctg ccc cct ccc gcg ccg gtg ccc tgc tgc tgc tgc ttc tgt tgc tga 96 SerAla Pro Ser Arg Ala Gly Ala Leu Leu Leu Leu Leu Leu Leu Leu 20 25 30 ggtctg cag gtt gct ggg gcg cag ggg aag ccc cgg ggg cgc tgt cca 144 Arg SerAla Gly Cys Trp Gly Ala Gly Glu Ala Pro Gly Ala Leu Ser 35 40 45 ctg ctgatc ccg ccg acc aga gcg tcc agt gtg tcc cca agg cca cct 192 Thr Ala AspPro Ala Asp Gln Ser Val Gln Cys Val Pro Lys Ala Thr 50 55 60 gtc ctt ccagcc ggc ctc gcc ttc tct ggc aga ccc cga cca ccc aga 240 Cys Pro Ser SerArg Pro Arg Leu Leu Trp Gln Thr Pro Thr Thr Gln 65 70 75 80 cac tgc cctcga cca cca tgg aga ccc aat tcc cag ttt ctg aag gca 288 Thr Leu Pro SerThr Thr Met Glu Thr Gln Phe Pro Val Ser Glu Gly 85 90 95 aag tcg acc catacc gct cct gtg gct ttt cct acg agc agg acc cca 336 Lys Val Asp Pro tyrArg Ser Cys Gly Phe Ser tyr Glu Gln Asp Pro 100 105 110 ccc tca ggg acccag aag ccg tgg ctc ggc ggt ggc cct gga tgg tca 384 Thr Leu Arg Asp ProGlu Ala Val Ala Arg Arg Trp Pro Trp Met Val 115 120 125 gcg tgc ggg ccaatg gca cac aca tct gtg ccg gca cca tca ttg cct 432 Ser Val Arg Ala AsnGly Thr His Ile Cys Ala Gly Thr Ile Ile Ala 130 135 140 ccc agt ggg tgctga ctg tgg ccc act gcc tga tct ggc gtg atg tta 480 Ser Gln Trp Val LeuThr Val Ala His Cys Leu Ile Trp Arg Asp Val 145 150 155 160 tct act cagtga ggg tgg gga gtc cgt gga ttg acc aga tga cgc aga 528 Ile tyr Ser ValArg Val Gly Ser Pro Trp Ile Asp Gln Met Thr Gln 165 170 175 ccg cct ccgatg tcc cgg tgc tcc agg tca tca tgc ata gca ggt acc 576 Thr Ala Ser AspVal Pro Val Leu Gln Val Ile Met His Ser Arg Tyr 180 185 190 ggg ccc agcggt tct ggt cct ggg tgg gcc agg cca acg aca tcg gcc 624 Arg Ala Gln ArgPhe Trp Ser Trp Val Gly Gln Ala Asn Asp Ile Gly 195 200 205 tcc tca agctca agc agg aac tca agt aca gca att acg tgc ggc cca 672 Leu Leu Lys LeuLys Gln Glu Leu Lys tyr Ser Asn tyr Val Arg Pro 210 215 220 tct gcc tgcctg gca cgg act atg tgt tga agg acc att ccc gct gca 720 Ile Cys Leu ProGly Thr Asp tyr Val Leu Lys Asp His Ser Arg Cys 225 230 235 240 ctg tgacgg gct ggg gac ttt cca agg ctg acg gca tgt ggc ctc agt 768 Thr Val ThrGly Trp Gly Leu Ser Lys Ala Asp Gly Met Trp Pro Gln 245 250 255 tcc ggacca ttc agg aga agg aag tca tca tcc tga aca aca aag agt 816 Phe Arg ThrIle Gln Glu Lys Glu Val Ile Ile Leu Asn Asn Lys Glu 260 265 270 gtg acaatt tct acc aca act tca cca aaa tcc cca ctc tgg ttc aga 864 Cys Asp AsnPhe tyr His Asn Phe Thr Lys Ile Pro Thr Leu Val Gln 275 280 285 tca tcaagt ccc aga tga tgt gtg cgg agg aca ccc aca ggg aga agt 912 Ile Ile LysSer Gln Met Met Cys Ala Glu Asp Thr His Arg Glu Lys 290 295 300 tct gctatg agc taa ctg gag agc cct tgg tct gct cca tgg agg gca 960 Phe Cys tyrGlu Leu Thr Gly Glu Pro Leu Val Cys Ser Met Glu Gly 305 310 315 320 cgtggt acc tgg tgg gat tgg tga gct ggg gtg cag gct gcc aga aga 1008 Thr Trptyr Leu Val Gly Leu Val Ser Trp Gly Ala Gly Cys Gln Lys 325 330 335 gcgagg ccc cac cca tct acc tac agg tct cct cct acc aac act gga 1056 Ser GluAla Pro Pro Ile tyr Leu Gln Val Ser Ser tyr Gln His Trp 340 345 350 tctggg act gcc tca acg ggc agg ccc tgg ccc tgc cag ccc cat cca 1104 Ile TrpAsp Cys Leu Asn Gly Gln Ala Leu Ala Leu Pro Ala Pro Ser 355 360 365 ggaccc tgc tcc tgg cac tcc cac tgc ccc tca gcc tcc ttg ctg ccc 1152 Arg ThrLeu Leu Leu Ala Leu Pro Leu Pro Leu Ser Leu Leu Ala Ala 370 375 380 tct1155 Leu 385 16 385 PRT Homo sapiens 16 Met Gly Arg Trp Cys Gln Thr ValAla Arg Gly Gln Arg Pro Arg Thr 5 10 15 Ser Ala Pro Ser Arg Ala Gly AlaLeu Leu Leu Leu Leu Leu Leu Leu 20 25 30 Arg Ser Ala Gly Cys Trp Gly AlaGly Glu Ala Pro Gly Ala Leu Ser 35 40 45 Thr Ala Asp Pro Ala Asp Gln SerVal Gln Cys Val Pro Lys Ala Thr 50 55 60 Cys Pro Ser Ser Arg Pro Arg LeuLeu Trp Gln Thr Pro Thr Thr Gln 65 70 75 80 Thr Leu Pro Ser Thr Thr MetGlu Thr Gln Phe Pro Val Ser Glu Gly 85 90 95 Lys Val Asp Pro Tyr Arg SerCys Gly Phe Ser Tyr Glu Gln Asp Pro 100 105 110 Thr Leu Arg Asp Pro GluAla Val Ala Arg Arg Trp Pro Trp Met Val 115 120 125 Ser Val Arg Ala AsnGly Thr His Ile Cys Ala Gly Thr Ile Ile Ala 130 135 140 Ser Gln Trp ValLeu Thr Val Ala His Cys Leu Ile Trp Arg Asp Val 145 150 155 160 Ile TyrSer Val Arg Val Gly Ser Pro Trp Ile Asp Gln Met Thr Gln 165 170 175 ThrAla Ser Asp Val Pro Val Leu Gln Val Ile Met His Ser Arg Tyr 180 185 190Arg Ala Gln Arg Phe Trp Ser Trp Val Gly Gln Ala Asn Asp Ile Gly 195 200205 Leu Leu Lys Leu Lys Gln Glu Leu Lys Tyr Ser Asn Tyr Val Arg Pro 210215 220 Ile Cys Leu Pro Gly Thr Asp Tyr Val Leu Lys Asp His Ser Arg Cys225 230 235 240 Thr Val Thr Gly Trp Gly Leu Ser Lys Ala Asp Gly Met TrpPro Gln 245 250 255 Phe Arg Thr Ile Gln Glu Lys Glu Val Ile Ile Leu AsnAsn Lys Glu 260 265 270 Cys Asp Asn Phe Tyr His Asn Phe Thr Lys Ile ProThr Leu Val Gln 275 280 285 Ile Ile Lys Ser Gln Met Met Cys Ala Glu AspThr His Arg Glu Lys 290 295 300 Phe Cys Tyr Glu Leu Thr Gly Glu Pro LeuVal Cys Ser Met Glu Gly 305 310 315 320 Thr Trp Tyr Leu Val Gly Leu ValSer Trp Gly Ala Gly Cys Gln Lys 325 330 335 Ser Glu Ala Pro Pro Ile TyrLeu Gln Val Ser Ser Tyr Gln His Trp 340 345 350 Ile Trp Asp Cys Leu AsnGly Gln Ala Leu Ala Leu Pro Ala Pro Ser 355 360 365 Arg Thr Leu Leu LeuAla Leu Pro Leu Pro Leu Ser Leu Leu Ala Ala 370 375 380 Leu 385

What is claimed:
 1. An isolated nucleic acid that specificallyhybridizes to DNA that is hypomethylated in cancer cells, said isolatednucleic acid comprising a sequence having greater than 90% homology toSEQ ID NO:15, said nucleic acid encoding a complete human tsp50 protein.2. The nucleic acid of claim 1 which comprises SEQ ID NO:15.
 3. Anisolated nucleic acid which encodes an amino acid sequence comprisingSEQ ID NO:16 from about amino acid residue number 1 to about amino acidresidue number
 385. 4. A recombinant DNA comprising the isolated nucleicacid of any one of claims 1 to 3 operably linked to regulatory controlsequences which effect expression of said nucleic acid in a host cell.5. An expression vector comprising the recombinant DNA of claim
 4. 6. Ahost cell comprising the expression vector of claim
 5. 7. The host cellof claim 6 wherein said host cell is a eukaryotic or prokaryotic cell.8. A process for producing a recombinant protein, which comprises: a)culturing the host cell of claim 6 in a culture medium under conditionssuitable for expression of said protein in said host cell, and b)recovering said protein from said host cell or said culture medium. 9.An oligonucleotide that specifically hybridizes to DNA that ishypomethylated in cancer cells, said oligonucleotide comprising asequence of at least about 12 adjacent nucleotides of SEQ ID NO:14 fromnucleotide number 1 to nucleotide number 705 or the complement of SEQ IDNO:14 from nucleotide number 1 to nucleotide number
 705. 10. Anoligonucleotide that specifically hybrizes to DNA that is hypomethylatedin cancer cells, said oligonuleotide comprising at least 15 adjacentnucleotides of SEQ ID NO:14 from nucleotide number 1 to nucleotidenumber 705 or the complement of SEQ ID NO:14 from nucleotide number 1 tonucleotide number
 705. 11. The oligonucleotide of claim 10 having SEQ IDNO:11.
 12. An oligonucleotide that specifically hybridizes to DNA thatis hypomethylated in cancer cells, said oligonucleotide comprising atleast 20 adjacent nucleotides of SEQ ID NO:14 from nucleotide number 1to nucleotide number 705 or the complement of SEQ ID NO:14 fromnucleotide number 1 to nucleotide
 705. 13. An isolated nucleic acid thatspecifically hybrizes to DNA that is hypomethylated in cancer cells,said isolated nucleic acid comprising SEQ ID NO:13 from about nucleotidenumber 1 to about nucleotide number 682 or the complement of SEQ IDNO:13 from about nucleotide number 1 to about nucleotide number
 682. 14.An isolated nucleic acid comprising SEQ ID NO:14 from about nucleotidenumber 1 to about nucleotide number 1240 or the complement of SEQ IDNO:14 from about nucleotide number 1 to about nucleotide number 1240.15. A kit for identifying whether genomic DNA in a test sample ishypomethylated or hypermethylated in a region which comprises TSP50 DNA,said kit comprising a nucleic acid hybridization assay probe thatspecifically hybridizes to DNA that is hypomethylated in cancer cells,and probe comprising SEQ ID NO:15 or its complement, or a sequencehaving greater than 90% homology to SEQ ID NO:15 or its complement thatencodes a complete human tsp50 protein and a restriction enzyme thatcleaves nonmethylated DNA but does not cleave methlated DNA.
 16. A kitfor identifying TSP50 mRNA in a test sample, said kit comprising anucleic acid hybridization assay probe that specifically hybridizes toDNA that is hypomethylated in cancer cells, said probe comprising SEQ IDNO:15 or a sequence having greater than 90% homolgy to SEQ ID NO:15 thatencodes a complete human tsp50 protein.
 17. A kit for identifying TSP50mRNA comprising oligonucleotide primers that specifically amplify cDNAsynthesized by reverse transcription of an mRNA from a test sample, saidcDNA comprising SEQ ID NO:15 or a sequence having greater than 90%homology to SEQ ID NO:15 that encodes a complete human tsp50 protein.18. The kit of claim 17 which comprises an oligonucleotide primerconsisting of having SEQ ID NO:11 and an oligonucleotide primerconsisting of SEQ ID NO:12.